Method of preparing an undifferentiated cell

ABSTRACT

Disclosed is a method of preparing an undifferentiated cell. The method includes contacting a more committed cell with an agent that causes the more committed cell to retrodifferentiate into an undifferentiated cell.

This application is a continuation-in-part of allowed U.S. applicationSer. No. 08/594,164, filed Jan. 31, 1996 now U.S. Pat. No. 6,090,625,and claiming priority from U.K. application No. 9502022.8, filed Feb. 2,1995, as well as a continuation-in-part of U.S. application Ser. No.09/521,700, filed Mar. 9, 2000 now abandoned as a division of U.S.application Ser. No. 08/594,164. Each of the foregoing application, andall documents cited in, or during the prosecution of each of theforegoing applications (“appln cited documents”) and each document filedby Applicant—either formally or informally—during the prosecution ofeach of the foregoing applications (“appln prosecution documents”), andeach document cited or referenced in each of the appln prosecutiondocuments (“prosecution document references”), and each document citedor referenced in each of the appln cited documents and in each of theprosecution document references, are hereby incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to a method of preparing anundifferentiated cell. In particular, the present invention relates to amethod of preparing an undifferentiated cell from a more committed cell.

In addition the present invention relates to the use of theundifferentiated cell of the present invention for the preparation of anew more committed cell—i.e. a recommitted cell.

The present invention also relates to the use of the undifferentiatedcell of the present invention or the recommitted cell of the presentinvention to have an effect (directly or indirectly via the use ofproducts obtained therefrom) on the immune system, such as thealleviation of symptoms associated with, or the partial or complete curefrom, an immunological condition or disease.

BACKGROUND TO THE INVENTION

Differentiation is a process whereby structures and functions of cellsare progressively committed to give rise to more specialised cells, suchas the formation of T cells or B cells from immature haemopoieticprecursors. Therefore, as the cells become more committed, they becomemore specialised. In the majority of mammalian cell types, celldifferentiation is a one-way process leading ultimately to terminallydifferentiated cells. However, although some cell types persistthroughout life without dividing and without being replaced, many celltypes do continue to divide during the lifetime of the organism andundergo renewal. This may be by simple division (e.g. liver cells) or,as in the case of cells such as haemopoietic cells and epidermal cells,by division of relatively undifferentiated stem cells followed bycommitment of one of the daughter cells to a programme of subsequentirreversible differentiation. All of these processes, however, have onefeature in common: cells either maintain their state of differentiationor become more differentiated. They do not become undifferentiated oreven less differentiated.

Retrodifferentiation is a process whereby structures and functions ofcells are progressively changed to give rise to less specialised cells.Some cells naturally undergo limited reverse differentiation(retrodifferentiation) in vivo in response to tissue damage. Forexample, liver cells have been observed to revert to an enzymeexpression pattern similar to the foetal enzymic pattern during liverregeneration (Curtin and Snell, 1983, Br. J. Cancer, Vol 48; 495-505).

Jose Uriel (Cancer Research, 1976, vol 36, pp 4269-4275) presented areview on the topic of retrodifferentiation, in which he said:

-   -   “retrodifferentiation appears as a common adaptive process for        the maintenance of cell integrity against deleterious agents of        varied etiology (physical, chemical, and viral). While        preserving the entire information encoded on its genome, cells        undergoing retrodifferentiation lose morphological and        functional complexity by virtue of a process of self-deletion of        cytoplasmic structures and the transition to a more juvenile        pattern of gene expression. This results in a progressive        uniformization of originally distinct cell phenotypes and to a        decrease of responsiveness to regulatory signals operational in        adult cells. Retrodifferentiation is normally counterbalanced by        a process of reontogeny that tends to restore the terminal        phenotypes where the reversion started. This explains why        retrodifferentiation remains invariably associated to cell        regeneration and tissue repair.”

Uriel (ibid) then went on to discuss cases of reportedretrodifferentiation—such as the work of Gurdon relating to nuclei fromgut epithelial cells of Xenopus tadpoles (Advances in Morphogenesis,1966, vol 4, pp 1-43. New York Academic Press, Eds Abercrombie andBracher), and the work of Bresnick relating to regeneration of liver(Methods in Cancer Research, 1971, vol 6, pp 347-391).

Uriel (ibid) also reported on work relating to isolated liverparenchymal cells for in vitro cultures. According to Uriel:

-   -   “Contrary to the results with fetal or neonatal hepatocytes,        with hepatocytes from regenerating liver, or from established        hepatomas, it has been difficult to obtain permanent class lines        from resting adult hepatocytes.”

Uriel (ibid) also reported on apparent retrodifferentiation in cancer,wherein he stated:

-   -   “the biochemical phenotypes of many tumours show analogous        changes of reversion toward immaturity . . . during the        preneoplastic phase of liver carcinogenesis, cells also        retrodifferentiate.”

More recent findings on retrodifferentiation include the work of MinoruFukunda (Cancer Research, 1981, vol 41: 4621-4628). Fukunda inducedspecific changes in the cell surface glycoprotein profile of K562 humanleukaemic cells by use of the tumour-promoting phorbol ester,12-O-tetradecanoyl-phorbol-13-acetate (TPA). According to Fukunda TPAappeared to induce the K562 human leukaemic cells into aretrodifferentiated stage.

Also, Hass et al. (Cell Growth & Differentiation, 1991, vol 2: 541-548)reported that long term culture of TPA-differentiated U-937 leukaemiacells in the absence of phorbol ester for 32-36 days resulted in aprocess of retrodifferentiation and that the retrodifferentiated cellsdetached from the substrate and reinitiated proliferation.

As mentioned above, another reported case of retrodifferentiation is thework of Curtin and Snell (Br. J. Cancer, 1983, vol 48: 495-505). Theseworkers compared enzymatic changes occurring duringdiethylnitrosamine-induced hepatocarcinogenesis and liver regenerationafter partial hepatectomy to normal liver differentiation. Thesesworkers found changes in enzyme activities during carcinogenesis thatwere similar to a step-wise reversal of differentiation. According tothese workers, their results suggest that an underlyingretrodifferentiation process is common to both the process ofhepatocarcinogenesis and liver regeneration.

More recently, Chastre et al. (FEBS Letters, 1985, vol 188 (2), pp2810-2811) reported on the retrodifferentiation of the human coloniccancerous subclone HT29-18.

Even more recently, Kobayashi et al. (Leukaemia Research, 1994, 18 (12):929-933) have reported on the establishment of a retrodifferentiatedcell line (RD-1) from a single rat myelomonocyticleukemia cell whichdifferentiated into a macrophage-like cell by treatment withlipopolysaccharide (LPS).

Much of the above prior art focuses on retrodifferentiation as a stagein carcinogenesis. Several prior art documents refer to experimentswhere tumour cell lines have apparently been retrodifferentiated.However, these prior art experiments were carried out using tumour celllines. The situation in genetically aberrant tumour cell lines is notcomparable with the normal differentiation pathways. Indeed, it isquestionable whether these results indicate true retrodifferentiation inthe sense of normal cell lineages. Further, the vast majority of theprior art retrodifferentiated cells were incapable of redifferentiatingto a more committed cell, whether of the same lineage, or of any otherlineage. One exception is given in Kobayashi et al., 1994, Vol 18;929-933 where a retrodifferentiated tumour cell line was differentiatedinto a macrophage-like cell using lipopolysaccharide. However, theretrodifferentiation achieved was very limited and the cells remainedcommitted both before and after treatment.

Similarly, the reverse differentiation seen to occur naturally in livercells is also very limited and can more accurately be classed asmodulations of the differentiated state, that is to say, reversiblechanges between closely related cell phenotypes.

SUMMARY OF THE INVENTION

Contrary to all earlier teachings, we have now shown that it is possibleto treat differentiated cells so that they become undifferentiatedcells, including stem cells. These undifferentiated cells are capable ofproliferating and giving rise to redifferentiated progeny of the samelineage or any other lineage. We believe that the process responsiblefor these changes is retrodifferentiation and thus we have nowsurprisingly found that it is possible to reverse the differentiationprocess in normal differentiated cells obtained from the human patientsto produce a stem cell. Furthermore, in the case of retrodifferentiatedhaematopoietic cells, these stem cells are pluripotent and can give riseto more than one cell lineage.

The clinical implications of this finding are enormous. Stem cells areextremely difficult to obtain from human patients. They are typicallyobtained from umbilical tissue, bone marrow or blood where they arepresent in only very small amounts. However, the present inventionprovides a method for producing stem cells from more committed cells bythe process of retrodifferentiation. Since more committed cells (such asB lymphocytes) are much more abundant in the human body, this techniqueprovides a powerful new method for obtaining stem cells. Thehaemopoietic stem cells exemplified in the present invention arepluripotent and are therefore capable of redifferentiating along morethan one cell lineage

Thus, according to a first aspect of the present invention there isprovided a method of preparing an undifferentiated cell, the methodcomprising contacting a more committed cell with an agent that causesthe more committed cell to retrodifferentiate into an undifferentiatedcell.

In a specific embodiment there is provided a method of increasing therelative number of undifferentiated cells in a cell population includingcommitted cells, which method comprises:

-   (i) contacting the cell population with an agent that operably    engages said committed cells; and-   (ii) incubating committed cells that are engaged by said agent such    that the relative number of undifferentiated cells increases as a    result of said engaging.

Preferably, the agent engages a receptor that mediates capture,recognition or presentation of an antigen at the surface of thecommitted cells. More preferably, the receptor is an MHC class I antigenor an MHC class II antigen, such as a class I antigen selected fromHuman-Leukocyte-Associated (HLA)-A receptor, an HLA-B receptor, an HLA-Creceptor, an HLA-E receptor, an HLA-F receptor or an HLA-G receptor or aclass II antigen selected from an HLA-DM receptor, an HLA-DP receptor,an HLA-DQ receptor or an HLA-DR receptor.

Typically, the committed cells are differentiated cells, preferablycells selected from T-cell colony-forming cells (CFC-T cells), B-cellcolony-forming cells (CFC-B cells), eosinophil colony-forming cells(CFC-Eosin cells), basophil colony-forming cells (CFC-Bas cells),granulocyte/monocyte colony-forming cells (CFC-GM cells), megakaryocytecolony-forming cells (CFC-MEG cells), erythrocyte burst-forming cells(BFC-E cells), erythrocyte colony-forming cells (CFC-E cells), T cellsand B cells.

In one preferred embodiment of the present invention, the more committedcell is not a cancer cell. In another preferred embodiment of thepresent invention, the agent is neither carcinogenic nor capable ofpromoting cancer growth.

In a preferred embodiment, the agent an antibody to the receptor, suchas a monoclonal antibody to the receptor. Specific examples includeCR3/43 and monoclonal antibody TAL.1B5.

Preferably the agent is used in conjunction with a biological responsemodifier, such as an alkylating agent, for example alkylating agent thatis or comprises cyclophosphoamide.

Preferred undifferentiated cells comprises a stem cell antigen. In apreferred embodiment, the undifferentiated cells are selected from anembryonic stem cell, a pluripotent stem cell, a lymphoid stem cell and amyeloid stem cell. Preferably, the undifferentiated cells arecharacterised by one or more of following cell surface markerdesignations: CD34⁺, HLA-DR⁻, CD38⁻ and/or CD45low. More preferably theundifferentiated cell is CD34⁺ and CD38⁻, even more preferably, CD34⁺,CD38⁻, HLA-DR⁻ and CD45low.

Thus in a preferred embodiment the present invention also provides amethod of increasing the relative number of cells having a cell surfacemarker designation CD34⁺, CD38⁻, HLA-DR⁻ and/or CD45low in a cellpopulation including committed cells, which method comprises:

-   (i) contacting the cell population with an agent that operably    engages said committed cells; and-   (ii) incubating committed cells that are engaged by said agent such    that the relative number of CD34⁺, HLA-DR⁻ and/or CD45low cells    increases as a result of said engaging.

In another embodiment, the present invention provides a method ofinducing committed cells in a cell population to become undifferentiatedcells capable of being recommitted into more differentiated cells whichmethod comprises:

-   (i) contacting the cell population with an agent that operably    engages said committed cells; and-   (ii) incubating committed cells that are engaged by said agent such    that they become undifferentiated cells as a result of said    engaging.

In a further embodiment, the present invention provides a method ofproducing an altered cell population comprising increased numbersundifferentiated cells capable of being recommitted into moredifferentiated cells, which method comprises:

-   (i) contacting an initial cell population comprising committed cells    with an agent that operably engages said committed cells; and-   (ii) incubating committed cells that are engaged by said agent such    that they become undifferentiated cells as a result of said    engaging, thereby resulting in an altered cell population comprising    increased numbers of said undifferentiated cells.

In any of the above methods, an optional step (iii) of enriching saidundifferentiated cells or recovering said undifferentiated cells fromthe altered cell population may be performed. Preferably, step (iii)comprises enriching said undifferentiated cells or recovering saidundifferentiated cells from the altered cell population by using a cellsurface marker present on the cell surface of the undifferentiated cellor a cell surface marker present on the surface of the committed cellsbut substantially absent from the cell surface of the undifferentiatedcells. Examples of suitable markers include CD34, CD45 and HLA-DR.

In another preferred embodiment, the undifferentiated cell of theinvention is CD34⁻ CD45⁻ and negative for markers of haemopoeiticlineages.

The undifferentiated cells produced by the methods of the presentinvention may be subsequently redifferentiated. Accordingly, the presentinvention provides a method of producing a committed/more differentiatedcell which method comprises contacting an undifferentiated cell producedby the methods of the invention with a compound that stimulatesdifferentiation of the undifferentiated cell. Suitable compounds includegrowth factors, colony stimulating factors and cytokines.

Thus according to a second aspect of the present invention there isprovided a method comprising contacting a more committed cell with anagent that causes the more committed cell to retrodifferentiate into anundifferentiated cell; and then committing the undifferentiated cell toa recommitted cell.

The term “recommitted cell” means a cell derived from theundifferentiated cell—i.e. a new more committed cell. “More committed”means more differentiated and can easily be determined by reference toknown pathways and stages of cell differentiation.

According to a third aspect of the present invention there is providedan undifferentiated cell produced according to the method of the presentinvention.

According to a fourth aspect of the present invention there is providedan undifferentiated cell produced according to the method of the presentinvention as or in the preparation of a medicament.

According to a fifth aspect of the present invention there is provided arecommitted cell produced according to the method of the presentinvention.

The more differentiated cells may be of the same lineage as the originalcommitted cells or of different lineage.

Thus as well as producing undifferentiated cells, the methods of thepresent invention can be used to convert cells of one lineage to thoseof another lineage. Accordingly, in a further aspect the presentinvention provides a method of inducing in a cell population comprisingcommitted hemopoietic cells of one hemopoietic lineage to become cellsof another hemopoietic lineage which method comprises:

-   (i) contacting the cell population with an agent that engages a    receptor that mediates capture, recognition or presentation of an    antigen at the surface of said committed hemopoietic cells; and-   (ii) incubating committed hemopoietic cells that are engaged by said    agent such that they become cells of another hemopoietic lineage as    a result of said engaging.

Preferably said committed cells are of a B cell lineage and become cellsof another hemopoietic lineage selected from a T cell lineage and amyeloid lineage.

Undifferentiated cells produced according to the methods of the presentinvention may be used to manufacture a medicaments for the treatment ofan immunological disorder or disease. Similarly, recommitted cellsproduced according to the methods of the present invention may be usedto manufacture a medicaments for the treatment of an immunologicaldisorder or disease.

Thus, in its broadest sense, the present invention is based on thehighly surprising finding that it is possible to form anundifferentiated cell from a more committed cell.

The present invention is highly advantageous as it is now possible toprepare undifferentiated cells from more committed cells and then usethose undifferentiated cells as, or to prepare, medicaments either invitro or in vivo or combinations thereof for the treatments ofdisorders.

The present invention is also advantageous as it is possible to committhe undifferentiated cell prepared by retrodifferentiation to arecommitted cell, such as a new differentiated cell, with a view tocorrecting or removing the original more committed cell or forcorrecting or removing a product thereof.

Preferably, the more committed cell is capable of retrodifferentiatinginto an MHC Class I⁺ and/or an MHC Class II⁺ undifferentiated cell.

Preferably, the more committed cell is capable of retrodifferentiatinginto an undifferentiated cell comprising a stem cell antigen.

Preferably, the more committed cell is capable of retrodifferentiatinginto a CD34⁺ undifferentiated cell.

Preferably, the more committed cell is capable of retrodifferentiatinginto a lymphohaematopoietic progenitor cell.

Preferably, the more committed cell is capable of retrodifferentiatinginto a pluripotent stem cell.

The findings presented herein may also be used to identify furtheragents that are capable of effecting retrodifferentiation of committedcells to undifferentiated cells. Accordingly, the present inventionprovides a method for identifying a substance capable ofretrodifferentiating a committed/differentiated cell to anundifferentiated cell, which method comprises contacting a population ofcells comprising committed cells with a candidate substance anddetermining whether there is an increase in the relative numbers ofundifferentiated cells in said cell population.

Preferably, said increase occurs within 24 hours, preferably 4 to 8hours (such that any changes cannot be solely accounted for by cellproliferation).

Typically, the determination of changes in the numbers ofundifferentiated cells is performed by monitoring changes in the numbersof cell having cell surface markers characteristic of undifferentiatedcells. Examples of suitable cell surface markers include CD34⁺.Alternatively, or in addition, decreases in the numbers of cells havingcell surface markers typical of differentiated cells and notundifferentiated cells may be monitored.

Preferably the committed cells used in the assay are committedhemopoietic cells such as cells selected from CFC-T cells, CFC-B cells,CFC-Eosin cells, CFC-Bas cells, CFC-GM cells, CFC-MEG cells, BFC-Ecells, CFC-E cells, T cells and B cells, more preferably B cells.

The present invention also provides an agent identified by the assaymethod of the invention and its use in a retrodifferentiation method ofthe invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts various haemopoietic cells

FIG. 2 is a scheme depicting differentiation pathways from lymphoid stemcells.

FIG. 3 is a scheme depicting differentiation pathways from myeloid stemcells.

FIG. 4 is a diagram of lymphohaemopoietic progenitor cells.

FIG. 5 is a scatter graph showing flow cytometry results.

FIG. 6 is a microscope picture of cells before treatment according tothe method of the present invention.

FIG. 7 is a microscope picture of cells prepared by the method of thepresent invention.

FIG. 8 is a microscope picture of cells prepared by the method of thepresent invention but at a lower magnification.

FIG. 9 is a microscope picture of cells before treatment according tothe method of the present invention.

FIG. 10 is a microscope picture of cells prepared by the method of thepresent invention.

FIG. 11 is a microscope picture of cells prepared by the method of thepresent invention.

FIG. 12A-B is a photomicrograph of a blood sample from a BCLL patientbefore treatment according to the method of the present invention. A andB are at different magnifications.

FIG. 13A-C is a photomicrograph of a blood sample from a BCLL patientduring treatment according to the method of the present invention

FIG. 14A-C is a photomicrograph of a blood sample from a BCLL patientduring treatment according to the method of the present invention

FIG. 15A-J is a photomicrograph of a blood sample from a BCLL patientafter treatment according to the method of the present invention,showing cells prepared by the method of the invention.

FIG. 16 is a photograph of a Southern blot.

FIG. 17A-B is (A) a photograph of an agarose gel containing PCR productsresolved by electrophoresis and stained with ethidium bromide and (B) aphotograph of a Southern blot.

FIG. 18 depicts scatter graphs showing flow cytometry results forhealthy cells treated with one of three different agents.

FIG. 19A-F is a photomicrograph showing the results of a colony formingassay conducted using purified normal B cells treated according to themethods of the invention.

FIG. 20 is a photomicrograph, using inverted bright field microscopy,showing the establishment of a long-term culture of stem cells(undifferentiated) produced according to the present invention (3 daysfollowing treatment).

FIG. 21 is a confocal microscopy image of cells before and aftertreatment according to the methods of the invention.

DETAILED DESCRIPTION OF THE INVENTION

I. Undifferentiated Cells and Differentiated Cell

There are many undifferentiated cells and differentiated cells found invivo and the general art is replete with general teachings on them.

By way of example, with respect to cells of the haempoietic celllineages, reference may be made to inter alia Levitt and Mertelsman 1995(Haematopoietic Stem Cells, published by Marcel Dekker Inc—especiallypages 45-59) and Roitt et al. (Immunology, 4th Edition, Eds. Roitt,Brostoff and Male 1996, Publ. Mosby—especially Chapter 10).

An undifferentiated cell is an immature cell that does not display amature differentiated character but is capable of yielding progeny thatdo. A well-known example of an undifferentiated cell is a stem cell.

Stem cells are undifferentiated immature cells, capable of self renewal(division without limit) and differentiation (specialisation). Thesejuvenile cells are abundant in a developing embryo, however, theirnumbers decrease as development progresses. By contrast, an adultorganism contain limited number of stem cells which are confined tocertain body compartments.

It is generally believed that stem cells are either monopotent, bipotentor pluripotent. Monopotent and bipotent stem cells are more restrictedin development and give rise to one or two types of specialised cells,respectively. In contrast, the pluripotent stem cells (PSCs) candifferentiate into many different types of cells, giving rise to tissue(which constitute organs) or in the case of totipotent stem cells, thewhole organism.

Pluripotent stem cells, unlike monopotent or bipotent, are capable ofmultilineage differentiation, giving rise to a tissue which wouldconsist of a collection of cells of different types or lineages.

According to the current understanding, as borne out by the teachingsfound on page 911 of Molecular Biology of the Cell (pub. GarlandPublishers Inc. 1983) and more recently Levitt and Mertelsman (ibid), astem cell, such as a pluripotent stem cell, has the following fourcharacteristics:

-   -   i. it is an undifferentiated cell—i.e. it is not terminally        differentiated;    -   ii. it has the ability to divide without limit;    -   iii. it has the ability to give rise to differentiated progeny;        and    -   iv. when it divides each daughter has a choice: it can either        remain as stem cell like its parent or it can embark on a course        leading irreversibly to terminal differentiation.

Note should be made of the last qualification, namely that according tothe general teachings in the art once an undifferentiated cell hasdifferentiated to a more committed cell it can not thenretrodifferentiate. This understanding was even supported by theteachings of Uriel (ibid), Fukunda (ibid), Hass et al (ibid), Curtin andSnell (ibid), Chastre et al (ibid), and Kobayashi et al (ibid) as theseworkers retrodifferentiated certain types of differentiated cells butwherein those cells remained committed to the same lineage and they didnot retrodifferentiate into undifferentiated cells.

Therefore, according to the state of the art before the presentinvention, it was believed that it was not possible to formundifferentiated cells, such as stem cells, from more committed cells.However, the present invention shows that this belief is inaccurate andthat it is possible to form undifferentiated cells from more committedcells.

The Haematopoietic Stem Cell is an example of a pluripotent stem cellwhich is found among marrow cells and gives rise to all the variousblood cells (including leucocytes and erythrocytes).

Blood is a fluid tissue, consisting of Lymphocyte (Ly), Monocytes (Mo),Neutrophils (Ne), Basophils (Ba), Eosinophils (Eso), Platelets (Pl) andRed Blood Cells (Rbc)—see FIG. 1. This specialised tissue is produced bythe differentiation of Haematopoietic Stem Cells (Hsc). In general, thewhite blood cells (inside blue circle) fight infections while red bloodcells (inside green circle) transport nutrients, oxygen and wasteproduct around the body.

Previously, haemopoietic stem cells were extracted by isolation from (i)bone marrow, (ii) growth factor mobilised peripheral blood or (iii) cordblood (placenta). Recently, haemopoietic stem cells have been preparedfrom Embryonic Stem Cells, which are extracted from embryos obtainedusing in vitro fertilisation techniques. These undifferentiated cellsare capable of multi-lineage differentiation and reconstitution of allbody tissue i.e. are totipotent.

The above mentioned extraction methods are cumbersome, sometimehazardous and in certain instances can be argued unethical, especially,in the case of the Embryonic Stem Cells extraction method.

There are a number of undifferentiated stem cells of the haemopoieticlineage. These include pluripotent stem cells (PSCs), lymphoid stemcells (LSCs) and myeloid stem cells (MSCs), known collectively aslymphohaematopoietic progenitor cells (LPCs). LSCs and MSCs are eachformed by the differentiation of PSCs. Hence, LSCs and MSCs are morecommitted than PSCs.

Examples of differentiated cells of the haemopoietic lineage include Tcells, B cells, eosinophils, basophils, neutrophils, megakaryocytes,monocytes, erythrocytes, granulocytes, mast cells, and lymphocytes.

T cells and B cells are formed by the differentiation of LSCs. Hence, Tcells and B cells are more committed than LSCs. In more detail, thechain of differentiation is LSC→pro-B-cell or prothymocyte.Pro-B-cell→pre-B-cell→mature B-cell→plasma cell. Prothymocyte→commonthymocyte→mature thymocytes (helper/inducer or cytotoxic/suppressorlineages)—see FIG. 2.

Eosinophils, basophils, neutrophils, megakaryocytes, monocytes,erythrocytes, granulocytes, mast cells, NKs, and lymphocytes are formedby the differentiation of MSCs. Hence, each of these cells are morecommitted than MSCs. In more detail, the chain of differentiation isMSC→immature megakaryoblast (→megakaryoblast→megakaryocyte→platelet) orproerythroblast (→erythroblast→reticulocyte→erythrocyte) ormyelomonocytic stem cell, a bipotent stem cell that differentiates toeither a myeloblast (→promyelocyt→myelocyt→granulocyte) or a monoblast(→promonocyte→monocyte→macrophage)—see FIG. 3.

The pathways of differentiation of haemotopoiesis have thus beenextensively characterised and the various cell stages are readilyidentifiable according to morphology and lineage-specific cell surfacemarkers (see below).

Other stem cells include neural stem cells, multipotent stem cells thatcan generate neurons, atrocytes and oligodendrocytes (Nakafuku andNakamura, 1995, J. Neurosci Res., vol 41(2): 153-68; Anderson, 1994,FASEB J., vol 8(10): 707-13; Morshead et al., 1994, Neuron, Vol 13(5):1071-82). Skeletal muscle satellite cells are another type of stem cell,more specifically a distinct class of myogenic cells that are maintainedas quiescent stem cells in the adult and can give rise to new musclecells when needed (Bischoff, 1986, Dev Biol., vol 115(1): 129-39). Othertypes of stem cells are epithelial stem cells, a subset of basal cells,and mesenchymal stem cells.

A very important type of stem cells are embryonic stem (ES) cells. Thesecells have been extensively studied and characterised. Indeed, ES cellsare routinely used in the production of transgenic animals. ES cellshave been shown to differentiate in vitro into several cell typesincluding lymphoid precursors (Potocnik et al., 1994, EMBO J., vol13(22): 5274-83) and neural cells. ES cells are characterised by anumber of stage-specific markers such as stage-specific embryonicmarkers 3 and 4 (SSEA-3 and SSEA-4), high molecular weight glycoproteinsTRA-1-60 and TRA-1-81 and alkaline phosphatase (Andrews et al., 1984,Hybridoma, vol 3: 347-361; Kannagi et al., 1983, EMBO J., vol 2:2355-2361; Fox et al., 1984, Dev. Biol., vol 103: 263-266; Ozawa et al.,1985, Cell. Differ., vol 16: 169-173).

Various antigens are associated with undifferentiated and differentiatedcells. The term “associated” here means the cells expressing or capableof expressing, or presenting or capable of being induced to present, orcomprising, the respective antigen(s).

Most undifferentiated cells and differentiated cells comprise MajorHistocompatability Complex (MHC) Class I antigens and/or Class IIantigens. If these antigens are associated with those cells then theyare called Class I⁺ and/or Class II⁺ cells.

Each specific antigen associated with an undifferentiated cell or adifferentiated cell can act as a marker. Hence, different types of cellscan be distinguished from each other on the basis of their associatedparticular antigen(s) or on the basis of a particular combination ofassociated antigens.

Examples of these marker antigens include the antigens CD34, CD19 andCD3. If these antigens are present then these particular cells arecalled CD34⁺, CD19⁺ and CD3⁺ cells respectively. If these antigens arenot present then these cells are called CD34⁻, CD19⁻ and CD3⁻ cellsrespectively.

In more detail, PSCs are CD34⁺DR⁻TdT⁻ cells (other useful markers beingCD38⁻ and CD36⁺). LSCs are DR⁺, CD34⁺ and TdT⁺ cells (also CD38⁺). MSCsare CD34⁺, DR⁺, CD13⁺, CD33⁺, CD7⁺ and TdT⁺ cells. B cells are CD19⁺,CD21⁺, CD22⁺ and DR⁺ cells. T cells are CD2⁺, CD3⁺, and either CD4⁺ orCD8⁺ cells. Immature lymphocytes are CD4⁺ and CD8⁺ cells. Activated Tcells are DR⁺ cells. Natural killer cells (NKs) are CD56⁺ and CD16⁺cells. T lymphocytes are CD7⁺ cells. Leukocytes are CD45⁺ cells.Granulocytes are CD13⁺ and CD33⁺ cells. Monocyte macrophage cells areCD14⁺ and DR⁺ cells. Additional details are provided in FIGS. 2 and 3.

Embryonic stem cells express SSEA-3 and SSEA-4, high molecular weightglycoproteins TRA-1-60 and TRA-1-81 and alkaline phosphatase. They alsodo not express SSEA-1, the presence of which is an indicator ofdifferentiation. Other markers are known for other types of stem cells,such as Nestein for neuroepithelial stem cells (J. Neurosci, 1985, Vol5: 3310). Mesenchymal stem cells are positive for SH2, SH3, CD29, CD44,CD71, CD90, CD106, CD120a and CD124, for example, and negative for CD34,CD45 and CD14.

Alternatively, or in addition, many cells can be identified bymorphological characteristics. The identification of cells usingmicroscopy, optionally with staining techniques is an extremely welldeveloped branch of science termed histology and the relevant skills arewidely possessed in the art. Clearly staining of cells will only becarried out on aliquots of cells to confirm identity since stains ingeneral cause cell death.

Hence, by looking for the presence of the above-listed antigen markersit is possible to identify certain cell types (e.g. whether or not acell is an undifferentiated cell or a differentiated cell) and thespecialisation of that cell type (e.g. whether that cell is a T cell ora B cell).

Undifferentiated cells may comprise any components that are concernedwith antigen presentation, capture or recognition. Preferably, theundifferentiated cell is an MHC Class I⁺ and/or an MHC Class II⁺ cell.

The more committed cell may comprise any components that are concernedwith antigen presentation, capture or recognition. Preferably, the morecommitted cell is an MHC Class I⁺ and/or an WIC Class II⁺ cell.

The more committed cell is any cell derived or derivable from anundifferentiated cell. Thus, in one preferred embodiment, the morecommitted cell is also an undifferentiated cell. By way of exampletherefore the more committed undifferentiated cell can be a lymphoidstem cell or a myeloid stem cell, and the undifferentiated cell is apluripotent stem cell.

In another preferred embodiment, the more committed cell is adifferentiated cell, such as a CFC-T cell, a CFC-B cell, a CFC-Eosincell, a CFC-Bas cell, a CFC-Bas cell, a CFC-GM cell, a CFC-MEG cell, aBFC-E cell, a CFC-E cell, a T cell, a B cell, an eosinophil, a basophil,a neutrophil, a monocyte, a megakaryocyte or an erythrocyte; and theundifferentiated cell is a myeloid stem cell, a lymphoid stem cell or apluripotent stem cell.

If the more committed cell is a differentiated cell then preferably thedifferentiated cell is a B lymphocyte (activated or non-activated), a Tlymphocyte (activated or non-activated), a cell from the macrophagemonocyte lineage, a nucleated cell capable of expressing class I orclass II antigens, a cell that can be induced to express class I orclass II antigens or an enucleated cell (i.e. a cell that does notcontain a nucleus—such as a red blood cell).

In alternative preferred embodiments, the differentiated cell isselected from any one of a group of cells comprising large granularlymphocytes, null lymphocytes and natural killer cells, each expressingthe CD56 and/or CD16 cell surface receptors.

The differentiated cell may even be formed by the nucleation of anenucleated cell.

II. Agents

The agent operably engages the more committed cell in order toretrodifferentiate that cell into an undifferentiated cell. In thisregard, the agent for the retrodifferentiation of the more committedcell into the undifferentiated cell may act in direct engagement or inindirect engagement with the more committed cell.

The agent may act intracellularly within the more committed cell.However, preferably, the agent acts extracellularly of the morecommitted cell.

An example of direct engagement is when the more committed cell has atleast one cell surface receptor on its cell surface, such as a β-chainhaving homologous regions (regions that are commonly found having thesame or a similar sequence) such as those that may be found on B cells,and wherein the agent directly engages the cell surface receptor.Another example, is when the more committed cell has a cell surfacereceptor on its cell surface such as an α-chain having homologousregions such as those that may be found on T cells, and wherein theagent directly engages the cell surface receptor.

An example of indirect engagement is when the more committed cell has atleast two cell surface receptors on its cell surface and engagement ofthe agent with one of the receptors affects the other receptor whichthen induces retrodifferentiation of the more committed cell.

The agent for the retrodifferentiation of the more committed cell intoan undifferentiated cell may be a chemical compound or composition.Preferably, however, the agent is capable of engaging a cell surfacereceptor on the surface of the more committed cell. Thus, in a preferredembodiment, the agent operably engages a receptor present on the surfaceof the more committed cell—which receptor may be expressed by the morecommitted cell, such as a receptor that is capable of being expressed bythe more committed cell.

For example, preferred agents include any one or more of cyclicadenosine monophosphate (cAMP), a CD4 molecule, a CD8 molecule, a partor all of a T-cell receptor, a ligand (fixed or free), a peptide, aT-cell receptor (TCR), an antibody, a cross-reactive antibody, amonoclonal antibody, or a polyclonal antibody. Growth factors may alsobe used, such as haemopoietic growth factors, for example erythropoietinand granulocyte-monocyte colony stimulating factor (GM-CSF).

If the agent is an antibody, a cross-reactive antibody, a monoclonalantibody, or a polyclonal antibody, then preferably the agent is any oneor more of an antibody, a cross-reactive antibody, a monoclonalantibody, or a polyclonal antibody to any one or more of: the β chain ofa MHC class II antigen, the β chain of a MHC HLA-DR antigen, the α chainof a MHC class I or class II antigen, the α chain of HLA-DR antigen, theα and the β chain of MHC class II antigen or of a MHC class I antigen.An example of a suitable antibody is CR3/43 (supplied by Dako).

The term “antibody” includes the various fragments (whether derived byproteolytic cleavage or recombinant technology) and derivatives thatretain binding activity, such as Fab, F(ab′)₂ and scFv antibodies, aswell as mimetics or bioisosteres thereof. Also included as antibodiesare genetically engineered variants where some of the amino acidsequences have been modified, for example by replacement of amino acidresidues to enhance binding or, where the antibodies have been made in adifferent species to the organism whose cells it is desired to treataccording to the methods of the invention, to decrease the possibilityof adverse immune reactions (an example of this is ‘humanised’ mousemonoclonal antibodies).

Agents used to effect the conversion of a more committed cell to anundifferentiated cell preferably act extracellularly of the morecommitted cell. In particular, it is preferred that the more committedcell comprises a receptor that is operably engageable by the agent andthe agent operably engages the receptor.

For example the receptor may be a cell surface receptor. Specificexamples of cell surface receptors include MHC class I and class IIreceptors. Preferably, the receptor comprises an α-component and/or aβ-component, as is the case for MHC class I and class II receptors.

More preferably, the receptor comprises a β-chain having homologousregions, for example at least the homologous regions of the β-chain ofHLA-DR.

Alternatively, or in addition, the receptor comprises an α-chain havinghomologous regions, for example at least the homologous regions of theα-chain of HLA-DR.

Preferably, the receptor is a Class I or a Class II antigen of the majorhistocompatibility complex (MHC). In preferred embodiments the cellsurface receptor is any one of: an HLA-DR receptor, a DM receptor, a DPreceptor, a DQ receptor, an HLA-A receptor, an HLA-B receptor, an HLA-Creceptor, an HLA-E receptor, an HLA-F receptor, or an HLA-G receptor. Inmore preferred embodiments the cell surface receptor is an HLA-DRreceptor.

Preferably, the agent is an antibody to the receptor, more preferablythe agent is a monoclonal antibody to the receptor.

Another preferred example of an agent is one that modulates MHC geneexpression such as MHC Class I⁺ and/or MHC Class II⁺ expression.

In a preferred embodiment, the agent is used in conjunction with abiological response modifier. Examples of biological response modifiersinclude an alkylating agent, an immunomodulator, a growth factor, acytokine, a cell surface receptor, a hormone, a nucleic acid, anucleotide sequence, an antigen or a peptide. A preferred alkylatingagent is or comprises cyclophosphoamide.

Other preferred biological response modifiers include compounds capableof upregulating MHC class I and/or class II antigen expression. In apreferred embodiment, this is so as to allow an agent that binds to anMHC receptor to work more effectively. Since any cell type can be madeto express MHC class I and/or class II antigens, this should provide amethod for retrodifferentiation a wide variety of cell types whetherthey constitutively express class I and/or class II MHC antigens or not.

III. Methods for Retrodifferentiating Cells

In the methods of the invention, a population of cells comprisingcommitted cells is contacted with an agent that operably engages one ormore committed cell in the population. The cell population is thenincubated so as to allow those cells that have been operably engaged bythe agent to progress through the retrodifferentiation process andultimately become undifferentiated.

Preferably the contacting step comprises the agent engaging with any oneor more of the following: homologous regions of the α-chain of class Iantigens, homologous regions of the α-chain of class II antigens, a CD4cell surface receptor, a CD8 cell surface receptor, homologous regionsof the β-chain of class II antigens in the presence of lymphocytes,homologous regions of the α-chain of class I antigens in the presence oflymphocytes, or homologous regions of the α-chain of class II antigensin the presence of lymphocytes. Preferably the contacting step occurs inthe presence of the biological response modifier (see above).

Typically, the population of cells is derived from a biological sample,such as blood or related tissues including bone marrow, neuronal tissuefrom the central nervous system or peripheral nervous system, or muscletissue. Preferably biological material is of post-natal origin. It ispreferred to use whole blood or processed products thereof, such asplasma, since their removal from subjects can be carried out with theminimum of medical supervision. Blood samples are typically treated withanticoagulents such as heparin or citrate. Cells in the biologicalsample may be treated to enrich certain cell types, remove certain celltypes or dissociate cells from a tissue mass. Useful methods forpurifying and separating cells include centrifugation (such as densitygradient centrifugation), flow cytometry and affinity chromatography(such as the use of magnetic beads comprising monoclonal antibodies tocell surface markers or panning). By way of example, Ficoll-Hypaqueseparation is useful for removing erythrocytes and granulocytes to leavemononuclear cells such as lymphocytes and monocytes.

Since the cells are essentially primary cultures, it may necessary tosupplement populations of cells with suitable nutrients to maintainviability. Suitable culture conditions are known by the skilled personin the art. Nonetheless, treatment of cell populations is preferablyinitiated as soon as possible after removal of biological samples frompatients, typically within 12 hours, preferably within 2 to 4 hours.Cell viability can be checked using well known techniques such as trypanblue exclusion.

Cell populations are generally incubated with an agent for at least twohours, typically between 2 and 24 hours, preferably between 2 and 12hours. Incubations are typically performed at from about roomtemperature, for example about 22° C., up to about 37° C. including 33°C. The progress of the retrodifferentiation procedure can be checkedperiodically by removing a small aliquot of the sample and examiningcells using microscopy and/or flow cytometry.

Once the relative numbers of the desired cell type have increased to asuitable level, which may for example be as low as 0.1% or as high as5%, the resulting altered cell populations may be used in a number ofways. With respect to the numbers of undifferentiated cells formed, itis important to appreciate the proliferative ability of stem cells.Although under some circumstance, the numbers of stem cells or otherundifferentiated cells formed may appear to be low, studies have shownthat only 50 pluripotent haemopoietic stem cells can reconstitute anentire haemopoietic system in a donor mouse. Thus therapeutic utilitydoes not require the formation of a large number of cells by the methodsof the invention.

Conversion of more committed cells to undifferentiated cells may also becarried out in vivo by administration of the agent, admixed with apharmaceutically carrier or diluent, to a patient. However it ispreferred in many cases that retrodifferentiation is performed invitro/ex vivo.

Treated populations of cells obtained in vitro may be used subsequentlywith minimal processing. For example they may be simply combined with apharmaceutically acceptable carrier or diluent and administered to apatient in need of stem cells.

It may however be desirable to enrich the cell population for theundifferentiated cells or purify the cells from the cell population.This can conveniently be performed using a number of methods. Forexample cells may be purified on the basis of cell surface markers usingchromatography and/or flow cytometry. Nonetheless, it will often beneither necessary nor desirable to extensively purify undifferentiatedcells from the cell population since other cells present in thepopulation (for example stromal cells) may maintain stem cell viabilityand function.

Flow cytometry is a well-established, reliable and powerful techniquefor characterizing cells within mixed populations as well as for sortingcells. Flow cytometry operates on the basis of physical characteristicsof particles in liquid suspension, which can be distinguished wheninterrogated with a beam of light. Such particles may of course becells. Physical characteristics include cell size and structure or, ashas become very popular in recent years, cell surface markers bound bymonoclonal antibodies conjugated to fluorescent molecules.

Kreisseg et al., 1994, J. Hematother 3(4): 263-89, state, “Because ofthe availability of anti-CD34 monoclonal antibodies, multiparameter flowcytometry has become the tool of choice for determination ofhaemapoietic stem and progenitor cells” and goes on to describe generaltechniques for quantitation and characterisation of CD34-expressingcells by flow cytometry. Further, Korbling et al., 1994, Bone MarrowTransplant. 13: 649-54, teaches purification of CD34⁺ cells byimmunoadsorption followed by flow cytometry based on HLA-DR expression.As discussed above, CD34⁺ is a useful marker in connection with stemcells/progenitor cells.

Flow cytometry techniques for sorting stem cells based on other physicalcharacteristics are also available. For example, Visser et al., 1980,Blood Cells 6:391-407 teach that stem cells may be isolated on the basisof their size and degree of structuredness. Grogan et al., 1980, BloodCells, 6: 625-44 also teach that “viable stem cells may be sorted fromsimple haemapoietic tissues in high and verifiable purity”.

As well as selecting for cells on the basis of the presence of a cellsurface marker or other physical property (positive selection), cellpopulations may be enriched, purified using negative criteria. Forexample, cells that possess lineage specific markers such as CD4, CD8,CD42 and CD3 may be removed from the cell population by flow cytometryor affinity chromatography.

A very useful technique for purifying cells involves the use ofantibodies or other affinity ligands linked to magnetic beads. The beadsare incubated with the cell population and cells that have a cellsurface marker, such as CD34, to which the affinity ligand binds arecaptured. The sample tube containing the cells is placed in a magneticsample concentrator where the beads are attracted to the sides of thetube. After one or more wash stages, the cells of interest have beenpartially or substantially completely purified from other cells. Whenused in a negative selection format, instead of washing cells bound tothe beads by discarding the liquid phase, the liquid phase is kept andconsequently, the cells bound to the beads are effectively removed fromthe cell population.

These affinity ligand-based purification methods can be used with anycell type for which suitable markers have been characterized or may becharacterized.

Urbankova et al., 1996. (J. Chromatogr B Biomed Appl. 687: 449-52)teaches the micropreparation of hemopoietic stem cells from a mouse bonemarrow suspension by gravitational field-flow fractionation. Urbankovaet al., 1996, further comments that the method was used for thecharacterization of stem cells from mouse bone marrow because thesecells are bigger than the other cells in bone marrow and it is thereforepossible to separate them from the mixture. Thus physical parametersother than cell surface markers may be used to purify/enrich for stemcells.

Cell populations comprising undifferentiated cells and purifiedundifferentiated cells produced by the methods of the invention may bemaintained in vitro using known techniques. Typically, minimal growthmedia such as Hanks, RPMI 1640, Dulbecco's Minimal Essential Media(DMEM) or Iscove's Modified Dulbecco Medium are used, supplemented withmammalian serum such as FBS, and optionally autologous plasma, toprovide a suitable growth environment for the cells. In a preferredembodiment, stem cells are cultured on feeder layers such as layers ofstromal cells (see Deryugina et al., 1993, Crit Rev. Immunology, vol 13:115-150). Stromal cells are believed to secrete factors that maintainprogenitor cells in an undifferentiated state. A long term culturesystem for stem cells is described by Dexter et al., 1977 (J. CellPhysiol, vol 91: 335) and Dexter et al., 1979 (Acta. Haematol., vol 62:299).

For instance, Lebkowski et al., 1992 (Transplantation 53(5): 1011-9)teaches that human CD34⁺ haemopoietic cells can be purified using atechnology based on the use of monoclonal antibodies that are covalentlyimmobilised on polystyrene surfaces and that the CD34⁺ cells purified bythis process can be maintained with greater than 85% viability.Lebkowski et al., 1993 (J. Hematother, 2(3): 339-42) also teaches how toisolate and culture human CD34⁺ cells. See also Haylock et al., 1994(Immunomethods, vol 5(3): 217-25) for a review of various methods.

Confirmation of stem cell identity can be performed using a number of invitro assays such as CFC assays (see also, the examples). Very primitivehaemopoietic stem cells are often measured using the long-term cultureinitiating cell (LTC-IC) assay (Eaves et al, 1991, J. Tiss. Cult. Meth.Vol 13: 55-62). LTC-ICs sustain haemopoiesis for 5 to 12 weeks.

Cell populations comprising undifferentiated cells and purifiedpreparations of comprising undifferentiated cells may be frozen forfuture use. Suitable techniques for freezing cells and subsequentlyreviving them are known in the art.

IV. Methods for Recommitting Undifferentiated Cells

One important application of undifferentiated cells of the presentinvention is in the reconstitution of tissues, for example nervoustissue or haemopoietic cells. This involves differentiating theundifferentiated cells produced by the methods of the invention. Thismay be carried out by simply administering the undifferentiated cells toa patient, typically at a specific site of interest such as the bonemarrow or spinal cord, and allowing the natural physiological conditionswithin the patient to effect differentiation. A specific example of thisis the reconstitution or supplementation of the haemopoietic system, forexample in the case of AIDS patients with reduced number of CD4⁺lymphocytes.

Alternatively, differentiation (also termed “recommitting”, herein) canbe effected in vitro and expanded cells then, for example, administeredtherapeutically. This is generally performed by administering growthfactors. For example, retinoic acid has been used to differentiate EScells into neuronal cells. Methylcellulose followed by co-culture with abone marrow stromal line and IL-7 has been used to differentiate EScells into lymphocyte precursors (Nisitani et al., 1994, Int. Immuno.,vol 6(6): 909-916). Bischoff, 1986 (Dev. Biol., vol 115(1): 129-39)teaches how to differentiate muscle satellite cells into mature musclefibres. Neural precursor cells can be expanded with basic fibroblastgrowth factor and epidermal growth factor (Nakafuku and Nakamura, 1995,J. Neurosci. Res., vol 41(2): 153-168). Haemopoietic stem cells can beexpanded using a number of growth factors including GM-CSF,erythropoeitin, stem cell factor and interleukins (IL-1, IL-3, IL-6)—seeMetcalf, 1989 (Nature, vol 339: 27-30) for a review of these variousfactors.

Potocnik et al., 1994 (EMBO J., vol 13(22): 5274-83) even demonstratedthe differentiation of ES cells to haemopoietic cells using low oxygen(5%) conditions.

Thus, in a preferred embodiment of the present invention theundifferentiated cell is then committed into a recommitted cell, such asa differentiated cell. The recommitted cell may be of the same lineageto the more committed cell from which the undifferentiated cell wasderived. Alternatively, the recommitted cell may be of a differentlineage to the more committed cell from which the undifferentiated cellwas derived. For example, a B lymphocyte may be retrodifferentiated to aCD34⁺CD38⁻HLA-DR⁻ stem cell. The stem cell may be subsequentlyrecommitted along a B cell lineage (the same lineage) or a lymphoidlineage (different lineage).

V. Assays for Identifying Retrodifferentiating Agents

In addition to the agents mentioned above, further suitable agents maybe identified using assay methods of the invention. Thus, the presentinvention provides a method for identifying a substance capable ofretrodifferentiating a committed/differentiated cell to anundifferentiated cell, which method comprises contacting a population ofcells comprising committed cells with a candidate substance anddetermining whether there is an increase in the relative numbers ofundifferentiated cells in said cell population.

Suitable candidate substances include ligands that bind to cell surfacereceptors such as antibody products (for example, monoclonal andpolyclonal antibodies, single chain antibodies, chimeric antibodies andCDR-grafted antibodies), such as antibodies that bind to cell surfacereceptors. Cell surface receptors of particular interest are describedabove and include MHC receptors and surface proteins of with CDdesignations, such as CD4 and CD8. Other ligands that bind to cellsurface receptors include growth factors.

Furthermore, combinatorial libraries, peptide and peptide mimetics,defined chemical entities, oligonucleotides, and natural productlibraries may be screened for activity as retrodifferentiation agents.The candidate substances may be used in an initial screen in batches of,for example 10 substances per reaction, and the substances of thosebatches which show inhibition tested individually.

A typical assay comprises placing an aliquot of cells comprisingcommitted cells in a suitable vessel such as a multiwell plate. Acandidate substance is added to the well and the cells incubated in thewell. Incubations are typically performed at from about roomtemperature, for example about 22° C., up to about 37° C. including 33°C.

Retrodifferentiation may be measured by removing a small aliquot ofcells and examining the cells by microscopy and/or flow cytometry todetermine whether there has been a change in the numbers ofundifferentiated cells. Typically, the determination of changes in thenumbers of undifferentiated cells is performed by monitoring changes inthe numbers of cell having cell surface markers characteristic ofundifferentiated cells, although morphological changes may also be usedas a guide. Examples of suitable cell surface markers include CD34⁺.Alternatively, or in addition, decreases in the numbers of cells havingcell surface markers typical of differentiated cells and notundifferentiated cells may be monitored, for example a reduction in therelative numbers of cells possessing lineage specific markers such asCD3, CD4 and CD8

Preferably, any increase in the numbers of cells having characteristicstypical of undifferentiated cells occurs within 24 hours, preferably 4to 8 hours, such that any changes cannot be solely accounted for by cellproliferation.

It may be desirable to prescreen for agents that bind to, for example,cell surface receptors, such as MHC class I or class II receptors. Anyagents identified as binding to target cell surface receptors may thenbe used in the above assay to determine their effect onretrodifferentiation. As a particular example, phage display librarieswhich express antibody binding domains may be used to identify antibodyfragments (typically scFvs) that bind to a target cell surface marker,such as the homologous region of the β-chain of MHC class II receptors.Suitable binding assays are known in the art, as is the generation andscreening of phage display libraries. Assays may also be used toidentify optimised antibodies or antibody fragments, for example toscreen a mutagenised library of derivatives of an antibody already shownto effect retrodifferentiation.

VI. Uses

The present invention provides methods of retrodifferentiating committedcells to undifferentiated cells. In particular, the present inventionprovides a method for preparing a stem cell from a more differentiatedcell. The clinical implications of this are enormous since stem cellsare being used in a wide variety of therapeutic applications but upuntil now were difficult, cumbersome and sometimes ethicallycontroversial to obtain.

Stem cells produced according to the present invention may be used torepopulate specific cell populations in a patient, such as ahaemopoietic cell population or a subpopulation thereof, such as CD4T-lymphocytes. The more committed cells used to produce the stem cellsmay be from the same patient or a matched donor. Thus stem cellsproduced according to the present invention may be used to heal andreconstitute specialised cell tissue and organs.

Thus, the present invention also encompasses a medicament comprising anundifferentiated cell prepared by any one of these processes admixedwith a suitable diluent, carrier or excipient.

In one embodiment, the medicament comprising the undifferentiated cellmay be used produce a beneficial more committed cell, such as one havinga correct genomic structure, in order to alleviate any symptoms orconditions brought on by or associated with a more committed cell havingan incorrect genomic structure. Thus, the present invention alsoprovides a process of removing an acquired mutation from a morecommitted cell wherein the method comprises forming an undifferentiatedcell by the method according to the present invention, committing theundifferentiated cell into a recommitted cell, whereby arrangement orrearrangement of the genome and/or nucleus of the cell causes themutation to be removed.

Preferably the gene is inserted into the immunoglobulin region or TCRregion of the genome.

The present invention also provides a method of treating a patientsuffering from a disease or a disorder resulting from a defective cellor an unwanted cell, the method comprising preparing an undifferentiatedcell by contacting a more committed cell with an agent that causes themore committed cell to retrodifferentiate into the undifferentiatedcell, and then optionally committing the undifferentiated cell into arecommitted cell; wherein the undifferentiated cell, or the recommittedcell, affects the defective cell or the unwanted cell to alleviate thesymptoms of the disease or disorder or to cure the patient of thedisease or condition.

Alternatively, the undifferentiated cell could be used to produce a morecommitted cell that produces an entity that cures any symptoms orconditions brought on by or associated with a more committed cell havingan incorrect genomic structure.

For example, the present invention may be used to prepare antibodies orT cell receptors to an antigen that is expressed by the more committedcell which has retrodifferentiated into the undifferentiated cell. Inthis regard, the antigen may be a fetospecific antigen or across-reactive fetospecific antigen.

The present invention also includes a process of controlling the levelsof undifferentiated cells and more committed cells. For example, thepresent invention includes a method comprising forming anundifferentiated cell by the method according to the present inventionand then activating an apoptosis gene to affect the undifferentiatedcell, such as bring about the death thereof.

In a preferred embodiment the present invention relates to a process ofintroducing a gene into the genome of an undifferentiated cell, whereinthe process comprises introducing the gene into a more committed cell,and then preparing an undifferentiated cell by the method according tothe present invention, whereby the gene is present in theundifferentiated cell.

In a more preferred embodiment the present invention relates to aprocess of introducing a gene into the genome of an undifferentiatedcell, wherein the process comprises inserting the gene into the genomeof a more committed cell, and then preparing an undifferentiated cell bythe method according to the present invention, whereby the gene ispresent in the undifferentiated cell.

The gene may be a gene that renders the undifferentiated cell and moredifferentiated cells obtained therefrom more resistant to pathogenicinfections such as a viral infection. In particular, by way of example,B lymphocytes from AIDS patients may be used to produce stem cells thatare then engineered to be resistant to HIV infection. When expanded andintroduced into the patients, the resulting helper T lymphocytes mayalso be resistant to HIV infection.

In an alternative embodiment the present invention relates to a processof introducing a gene into an undifferentiated cell, wherein the processcomprises inserting the gene into the genome of a more committed cell,and then preparing an undifferentiated cell by the method according tothe present invention, whereby the gene is present in the genome of theundifferentiated cell.

In addition, the present invention also encompasses the method of thepresent invention for preparing an undifferentiated cell, wherein themethod includes committing the undifferentiated cell into a recommittedcell and then fusing the recommitted cell to a myeloma. This allows theexpression in vitro of large amounts of the desired product, such as anantibody or an antigen or a hormone etc.

The present invention encompasses an undifferentiated cell prepared byany one of these processes of the present invention.

Other aspects of the present invention include:

The use of any one of the agents of the present invention for preparingan undifferentiated cell from a more committed cell.

The use of an undifferentiated cell produced according to the method ofthe present invention for producing any one of a monoclonal or apolyclonal or a specific antibody from a B-lymphocyte or a T-lymphocyte;a cell from the macrophage monocyte lineage; a nucleated cell capable ofexpressing class I or class II antigens; a cell capable of being inducedto express class I or class II antigens; an enucleated cell; afragmented cell; or an apoptic cell.

The use of an undifferentiated cell produced according to the method ofthe present invention for producing effector T-lymphocytes fromB-lymphocytes and/or vice versa.

The use of an undifferentiated cell produced according to the method ofthe present invention for producing any one or more of: a medicament,such as a medicament comprising or made from a B-lymphocyte, aT-lymphocyte, a cell from the macrophage monocyte lineage, a nucleatedcell capable of expressing a class I or a class II antigen, a cellcapable of being induced to express a class I or a class II antigen, oran enucleated cell.

The present invention also encompasses processes utilising theafore-mentioned uses and products or compositions prepared from suchprocesses.

The present invention also encompasses a medicament comprising anundifferentiated cell according to the present invention or a productobtained therefrom admixed with a suitable diluent, carrier orexcipient.

In one preferred embodiment the medicament comprises an antibody orantigen obtained from an undifferentiated cell according to the presentinvention admixed with a suitable diluent, carrier or excipient.

Preferably the medicament is for the treatment of any one of: cancer,autoimmune diseases, blood disorders, cellular or tissue regeneration,organ regeneration, the treatment of organ or tissue transplants, orcongenital metabolic disorders.

The methods of the invention and products obtained by those methods,such as undifferentiated cells, may be used in research, for example tostudy retrodifferentiation, differentiation and identify and study newdevelopmental antigens and cluster differentiation antigens.

VII. Administration

Stem cells and recommitted cells of the present invention, as well asagents shown to retrodifferentiate cells, may be used in therapeuticmethods. Preferably the cells or agents of the invention are combinedwith various components to produce compositions of the invention. Morepreferably the compositions are combined with a pharmaceuticallyacceptable carrier or diluent to produce a pharmaceutical composition(which may be for human or animal use). Suitable carriers and diluentsinclude isotonic saline solutions, for example phosphate-bufferedsaline. The composition of the invention may be administered by directinjection. The composition may be formulated for parenteral,intramuscular, intravenous, subcutaneous, intraocular, oral ortransdermal administration.

Compositions comprising cells are typically delivered by injection orimplantation. Cells may be delivered in suspension or embedded in asupport matrix such as natural and/or synthetic biodegradable matrices.Natural matrices include collagen matrices. Synthetic biodegradablematrices include polyanhydrides and polylactic acid. These matricesprovide support for fragile cells in vivo and are preferred fornon-haemopoetic cells.

Delivery may also be by controlled delivery i.e. over a period of timewhich may be from several minutes to several hours or days. Delivery maybe systemic (for example by intravenous injection) or directed to aparticular site of interest.

Cells are typically administered in doses of from 1×10⁵ to 1×10⁷ cellsper kg. For example a 70 kg patient may be administered 14×10⁶ CD34⁺cells for reconstitution of haemopoietic tissues.

The routes of administration and dosages described are intended only asa guide since a skilled practitioner will be able to determine readilythe optimum route of administration and dosage for any particularpatient and condition.

The present invention will now be described by way of examples, whichare illustrative only and non-limiting.

A. Materials and Methods

Patients

Blood samples were obtained in lavender top tubes containing EDTA frompatients with B-cell chronic lymphocytic leukaemias, patients withantibody deficiency (including IgA deficiency and X-linked infantilehypogammaglobulinaemias), patients with HIV infections and AIDSsyndrome, a patient with CMV infection, a patient with Hodgkin'slymphomas, a patient with acute T-cell leukaemia, a 6-days old baby withblastcytosis, various patients with various infections and clinicalconditions, cord blood, bone marrow's, and enriched B-lymphocytepreparations of healthy blood donors.

Clinical and Experimental Conditions

The clinical and experimental treatment conditions of patients,including various types of treatment applied to their blood samples, aredescribed in Table 1. Differential white blood cell (WBC) counts wereobtained using a Coulter Counter and these are included in the sameTable.

Treatment of Blood

Blood samples, once obtained, were treated with pure monoclonal antibodyto the homologous region of the β-chain of the HLA-DR antigen (DAKO) andleft to mix on a head to head roller at room temperature for a maximumof 24 hours. Some samples were mixed first on a head to head roller for15 minutes after which they were left to incubate in an incubator at 22°C. The concentration of monoclonal antibody added to blood samplesvaried from 10-50 μl/ml of blood.

In addition, other treatments treatments were applied at the sameconcentrations and these included addition of a monoclonal antibody tothe homologous of the α-chain of the HLA-DR antigen, a monoclonalantibody to the homologous region of class I antigens, a monoclonalantibody to CD4, a monoclonal antibody to CD8, and a PE conjugatedmonoclonal antibody to the homologous region of the β-chain of theHLA-DR antigen.

Other treatments included the simultaneous addition of monoclonalantibodies to the homologous regions of the α and β-chains of the HLA-DRantigen to blood samples.

Furthermore, alkylating agents such as cyclophosphoamide were added toblood samples in combination with pure monoclonal antibody to thehomologous region of the β-chain of the HLA-DR antigen.

Following these treatments blood samples were stained with panels oflabelled monoclonal antibodies as instructed by the manufacturer'sinstructions and then analyzed using flow cytometry.

Incubation periods with monoclonal antibodies ranged from 2 hour, 4hour, 6 hour, 12 hour to 24 hour intervals.

Labelled Antibodies

The following monoclonal antibodies were used to detect the followingmarkers on cells by flow cytometry: CD19 and CD3, CD4 and CD8, DR andCD3, CD56 & 16 and CD3, CD45 and CD14, CD8 and CD3, CD8 and CD28,simultest control (IgG1 FITC+IgG2a PE), CD34 and CD2, CD7 and CD13 & 33,CD10 and CD25, CD5 and CD10, CD5 and CD21, CD7 and CD5, CD13 and CD20,CD23 and CD57 and CD25 and CD45 RA (Becton & Dickenson and DAKO).

Each patient's blood sample, both treated and untreated, was analyzedusing the majority of the above panel in order to account for theimmunophenotypic changes that accompanied different types of treatmentsand these were carried out separately on different aliquots of the sameblood sample. Untreated samples and other control treatments werestained and analyzed simultaneously.

Flow Cytometry

Whole blood sample was stained and lysed according to the manufacturerinstructions. Flow cytomery analysis was performed on a FACScan@ witheither simultest or PAINT A GATE software (BDIS) which included negativecontrols back tracking. 10,000 to 20,000 events were acquired and storedin list mode files.

Morphology

Morphology was analyzed using microscopy and Wright's stain.

Preparing Stem Cells from Enriched or Purified B-CLL (or Normal)Lymphocytes:

Aseptic techniques should be used throughout the following procedures:

(A) Mononuclear Cell Separation:

(i) Obtain mononuclear cells from peripheral blood samples bycentrifugation on Histopaque, Lymphoprep, or any Lymphocyte separationmedium (sp. gray 1.077) for 30 mins at 400 g.

(ii) Collect mononuclear cells in a 50 ml conical tube and wash with 30mls of Hank's balanced salt solution (Ca²⁺ and Mg⁺ free, Sigma)containing 2% FCS and 2 mM EDTA or 0.6% citrate.

(iii) After washing, count cells and assess viability using trypan blueand haemocytometer.

(iv) If B-cell count is high, above 70% (20×10⁹/L, WBC), proceedstraight to B (vi).

(v) If B-cell count is low, below 70% (20×10⁹/L, WBC). Perform negativeselection using Macs microbeads or FacsVantage purification technique,as described below in Section 1. C.

(vi) Resuspend cell pellet at a concentration of 3×10⁶/ml in IMDM medium(100 μg/ml streptomycin), containing 10% FCS (heat inactivated) and 10%HS (heat inactivated). Note: If no FCS and HS available, use 20% to 50%autologous plasma.

(B) Cell Treatment Using Pure CR3/43 (Dako) Monoclonal Antibody:

After mononuclear cell separation has been achieved in A (vi), proceedwith the following:

(i) Use a culture tray with six wells, add 2 mls of cell suspension[from A (vi) above] to each well of this multi-well culture tray.

(ii) Treat five wells each with 7.5 μl/ml of CR3/43—(pure monoclonalantibody, Dako) and leave one well untreated (negative control.

(iii) Incubate the culture tray in 5% CO₂ at 37 C.°.

(C) Purification of Cells:

Negative Selection of B cells using MACS microbeads (Miltenyi Biotec,here it is best to follow manufacturer instructions):

(i) Obtain mononuclear cells as in Section A above.

(ii) Pellet and resuspend cells in a final volume of 300 μl per 10⁸total cells in HBSS (consisting of 2% FCS and 2 mM EDTA or 0.6%citrate).

(iii) Add 100 μl per 10⁸ total cells of pure monoclonal antibody to CD2(IgG1, DAKO).

(iv) To the same cell suspension add 50 μl per 10⁸ per total cells ofpure monoclonal antibody to CD33 (IgG1, DAKO).

(v) Leave the mixture to incubate for 10 minutes at room temperature.

(vi) Wash cells with HBSS (containing 2% FCS and 2 mM EDTA) andresuspend at a final concentration of 400 μl per 10⁸ total cells, withthe same buffer.

(vii) Add 100 μl of rabbit anti-mouse IgG1 labelled microbeads per 10⁸total cells (or follow manufacturer instructions).

(viii) Thoroughly mix cells and incubate at 6 C.° to 12 C.° (fridge) for15 minutes.

(ix) Again wash cells with HBSS (containing 2% FCS and 2 mM EDTA) andresuspend at a final concentration of 500 μl per 10⁸ total cells, withthe same buffer.

(x) Assemble MS+/RS+ column in the magnetic field of the MACS separator.

(xi) Wash column with 3 mls of HBSS (containing 2% FCS and 2 mM EDTA).

(xii) Pass cells through column and then wash with 4×500 μl with HBSS(containing 2% FCS and 2 mM EDTA).

(xiii) Elute and collect cells in a conical tube, then pellet andresuspended in IMDM as in Section A (vi).

D) FACSVantage Purified B Cells:

(i) Obtain mononuclear cells from peripheral blood samples of B-CLLpatients, as described in Section A, above.

(ii) Stain these cells with a combination of CD19-PE and CD20-FITCconjugated monoclonal antibodies to identify the B cells.

(iii) On the basis of CD19/CD20 fluorescence, sort approximately 10⁷cells using a Beckton Dickenson FACS Vantage and argon laser emitting at488 nm.

(iv) Wash purified cells with Hanks balanced salt solution containing50% FCS and then allow to recover overnight at 37° C. in a humidifiedincubator at 5% CO₂.

(v) Pellet and resuspend cells as described in Section A (vi) above andthen treat with CR3/43 as described in Section B above.

Preparing Stem Cells in Whole Blood Cells

Treatment of cells with pure CR3/43 (Dako) monoclonal antibody in wholeblood:

(i) Select patients with WBC counts of 30-200×10⁹/L (ranging from 73-95%B lymphocytes).

(ii) Collect blood by venipuncture into citrate, EDTA- or preservativefree heparin containing tubes.

(iii) Add CR3/43 Antibody directly to whole blood, at a finalconcentration of 0.08-0.16 μg/10⁶ cells (e.g. if WBC count was 50×10⁹/Lthen 50 μl of CR3/43 monoclonal antibody, mouse IgG concentration of 159μg/ml, should be added per ml of blood).

(iv) Mix blood thoroughly and leave overnight at room temperature in anincubator.

(v) Analyse blood cells 0 hr, 2 hr, 6 hr and 24 hr after the addition ofmAbs.

Note: Due to the homotypic aggregation of B cells and the formation ofadherent cells in the bottom of the test tube, induced by mAb CR3/43,thoroughly mix and sample cells using wide-bore pipette tips or 21-Gneedle before analysis.

In order to obtain a uniform population of cells throughout theanalysis, divide blood sample into separate aliquots prior CR3/43treatment.

Preparation for Analysis of Stem Cells Produced by Treating Cultured BCells with CR3/43 Monoclonal Antibody.

Stem cells produced using the methods of the invention can be assessedat a number of times points, for example every 2 hr, 7 hr, 24 hr, daily,7 days or longer periods (months, following weekly feeding of cells withlong term culture medium). In order to analyse all cells in the wellincluding adherent and non-adherent layer, together or separately, onewell has to be sacrificed.

(i) Gently remove non-adherent layer using a wide bore pipette anddisrupt cell clumps by repeated aspiration through 21-G needle to obtainsingle cell suspension.

(ii) Using a cell scraper scrape adherent layer and disrupt gently cellclumps to obtain single cell suspension by repeated aspiration through a21-G needle.

(iii) Alternatively, trypsinize adherent layer by first rinsing withHBSS and then adding 2 ml of 0.25% trypsin per well and incubate at 37C.° for 10 minutes.

(iv) Gently disturb cell clumps by repeated pipetting.

(v) After 10 mins incubate with 20% of FCS to a final concentration toinactivate the trypsin.

(vi) Wash cells twice with IMDM and 2% FCS by centrifugation at 800 gfor 10 mins.

(vii) Count cells and assess viability.

Analysis of Stem Cells:

The following methods can be used for the assessment of stem cells.

(A) Immunophenotype:

For Immunophenotypic analysis (using Flow Cytometry).

(i) For whole blood samples, immunostain (according to manufacturerinstructions), lyse the erythrocytes and wash the cells after theincubation period and treat with mAb. Lysing and wash solutions fromBecton Dickinson may typically be used.

(ii) Leukocytes (in whole blood, mononuclear fraction, MACS microbeadsnegatively selected B cells or sorted B-CLL) should be either doubly orsingly labelled with mAbs conjugated directly to fluoresceinisothiocyanate (FITC) or phycoerythrin (PE).

(iii) Perform double labelling using IMK+ kit (Becton Dickinson):consisting of the following monoclonal antibody pairs:

-   -   CD45-FITC and CD14-PE;    -   CD19-PE and CD3-FITC;    -   CD8-PE and CD4-FITC;    -   HLA-DR-PE and CD3-FITC; and    -   CD56, CD16-PE and CD3-FITC.

Also isotype match negative controls for IgG₁-FITC and IgG_(2a)-PE areincluded.

(iv) The following additional antibodies can also be used which aremanufactured by Dako and Becton Dickinson:

PE-conjugated: anti-CD8, anti-CD33, anti-13, anti-CD34, anti-CD19,anti-CD2, anti-CD14, anti-CD33 and anti-CD5;

FITC-conjugated: anti-CD3, anti-CD7, anti-IgM, anti-CD22, anti-CD20,anti-CD10, anti-CD7, anti-CD16, anti-TCRαβ.

(v) The following can also be used.

-   -   Also affinity purified IgG₃ mAb specific for CD34 (Dako) can be        used and is detect with FITC- or PE-labelled goat anti-mouse        immunoglobulin F(ab)′₂ fragment as secondary antibody (DAKO).    -   Quantum Red (PE-Cy5)-conjugated anti-CD34 (Dako) was also used.

(vi) Analyse cells using FACScan or FACS Vantage (Becton Dickinson).

(vii) Analyse data using Proprietary Paint-a-Gate, Lysis II, Consort 30and CellQuest software.

(B) Morphology:

For morphological analysis:

Light Microscopy

(i) Resuspend cells thoroughly using wide-bore pipette tips or 21-Gneedle.

(ii) Examine under a Leitz microscope using Wright's or Giemsa stains.

(iii) Morphological analysis of B-CLL lymphocytes can be performed inblood films or cytocentrifuged preparations, respectively.

Confocal Microscopy

(i) Obtain B cells as described above (B-CLL or healthy B cells obtainedfrom buffy coat of healthy blood donors)

(ii) Treat B cells with CR3/43 monoclonal antibody as described above.

(iii) Add 2 ml of cell suspension to an organ culture dish (The bottomof this dish is engineered to have a cover-slip).

(iv) Add 15 μl of monoclonal antibody to CD19 FITC-conjugate and 15 μlof monoclonal antibody to CD34 PE/Cy 5-conjugate (Quantum Red).

(v) Use Propidium Iodide to assess viability and Hoechst to stain thenuclei.

(C) PCR Analysis of VDJ Gene Rearrangement

The VDJ region of the IgH gene was analyzed by PCR (Perkin elmer thermalcycler) using template DNA from B-CLL peripheral blood samples beforeand after (2 hr, 6 hr and 24 hr) antibody treatment. The β-actin genewas used as a control. For the VDJ region, V_(H)1, V_(H)2, V_(H)3,V_(H)4, V_(H)5 and V_(H)6 family-specific sense primers were used with Jantisense primers (Deane and Norton, 1990). All primers were synthesizedby the Molecular Biology Unit, Randall Institute, King's College,London.

(D) Southern Analysis of VDJ Gene Rearrangement

(i) Digest the Genomic DNA from treated and untreated peripheral bloodsamples or purified B cells (from B-CLL patients), usingBamHI/HindII—typically cells from a number of wells are required to givea sufficient amount of DNA to conduct the analysis.

(ii) The digests were resolved on 0.8% agarose gels and transferred toGeneScreen® nylon membranes (Dupont) according to manufacturer'sinstructions (Southern, 1975).

(iii) The rearrangement of the IgH gene can be characterised byanalysing the J region of the IgH locus, using ³²P-labeled human J_(H)DNA probe isolated from placental genomic DNA (Calbiochem, OncogeneScience).

(iv) Autoradiographs should be kept at −70° C. for several days prior todeveloping.

(E) Long Term Culture:

Cell cultures prepared as described above can be maintained for longerperiods (long term culture) by weekly feeding using long term culturemedium (IMDM, 10% FCS, 10% HS, 1% hydrocortisone 5×10⁻⁷M stocksolution).

(i) First, following 24 hr from the initiation of CR3/43 treatmentdilute cells in each well by adding 2 mls of long term culture medium.

(ii) Feed wells weekly following removal of half of the growth medium.

(iii) Inspect wells using phase-contrast microscopy.

(F) Colony Forming Assays:

(i) After, 24 hr following initiation (or longer incubation period withpure monoclonal antibody CR3/43) of treatment, obtain 300 μl in culturemedium of the non-adherent cells as described above.

(ii) Add to the cell suspension in the culture medium above, 3 mls ofmethocult GFH4434 (StemCell Technologies, consisting of methylcellulosein Iscove's MDM, FCS, BSA, L-glutamine, rh stem cell factor, rh GM-CSF,rh IL-3 and rh erythropoietin).

(iii) Take 1.1 ml of cell mixture and plate in triplicate.

(iv) Incubate the plates at 37° C. in a humidified petri dish with 5%CO₂ and 5% O2 for 14 days.

(v) Inspect the wells before and after treatment with CR3/43 monoclonalantibody using phase-contrast microscopy.

B. Results

CD19 and CD3 Panel

Treatment of blood samples with monoclonal antibody to the homologousregion of the β-chain of the HLA-DR antigen always decreased therelative number of CD19⁺ cells. This marker is a pan B-cell antigen (seeTables). This antigen is present on all human B lymphocytes at allstages of maturation but is lost on terminally differentiated plasmacells. Hence, this is an indication that B cells wereretrodifferentiating into undifferentiated cells.

The same treatment caused the relative number of CD3⁺ cells to increasedramatically especially in blood of patients with B-CLL, which wasalways accompanied by an increase in the relative number in CD3⁻CD19⁻cells. CD3 is present on all mature T-lymphocytes and on 65%-85% ofthymocytes. This marker is always found in association with α-/β- orgamma/delta T-cell receptors (TCR) and together these complexes areimportant in transducing signals to the cell interior. Hence, this is anindication that B cells were retrodifferentiating into undifferentiatedcells and then being committed to new differentiated cells, namely Tcells.

A novel clone of cells appeared in treated blood of B-CLL patientsco-expressing the CD19 and CD3 markers—i.e. CD19⁺ and CD3⁺ cells (seeChart 1, patient 2, 3 & 4 at 2 hr, 6 hr & 24 hr of starting treatment).Other patients with different conditions showed an increase in therelative number of these clones of cells. These cells were exceptionallylarge and heavily granulated and extremely high levels of CD19 wereexpressed on their cell membrane. The CD3 marker seems to be expressedon these cells at similar levels to those expressed on normal maturelymphocytes.

In Table 2, patient numbers 2, 3 and 4 are actually numbers representingthe same patient and their delineation was merely to show the effect oftreatment on blood with time (See Table 1 for experimental and clinicalcondition of this patient).

The CD19⁺CD3⁺ clones in treated samples seem to decrease with time,reaching original levels to those determined in untreated sample at 2hrs, 6 hrs and 24 hrs.

Another type of cell of the same size and granulity was detected intreated samples and these cells had high levels of CD19 expressed ontheir surface but were negative for the CD3 marker and rich in FCreceptors. However, the relative number of these cells appeared todecrease in time. Of interest, at 24 hours treatment of blood sample (2,3 and 4) there was a decrease in the relative number of CD19⁻CD3⁻ cellsin a group of cells that were initially observed to increase after 2 and6 hrs treatment of blood samples. However, Coulter counts of WBCpopulations were reduced on treatment of blood with monoclonal antibodyto the homologous region of the β-chain of the HLA-DR antigen. Thisfinding suggests that this type of treatment gives rise to atypicalcells that cannot be detected by Coulter (Table 1) but can be accountedfor when measured by flow cytometry which counts cells on the basis ofsurface markers, size and granulity. Furthermore, these atypical cellswere accounted for by analysing morphology using Wright's stain under amicroscope. Flow cytometric charts of these phenomena are represented inCharts (1, 2, 3 & 4) and the immunophenotypic changes obtained ontreatment of blood samples seems to suggest that CD19⁺ and CD3⁺lymphocytes are an interconnected group of cells but remain distinct onthe basis of CD19 and CD3 relative expression compared to stem cells.

In Table 2, patient numbers 5 and 6 represent the same patient butanalysis of treated and untreated blood samples were monitored with timeand at the same time (see Table 1).

Patients' blood with no B-cell malignancy showed similar trends ofimmunophenotypic changes when compared to blood of B-CLL patients butthe changes were not to the same extent. However, the relative andabsolute number of B-lymphocytes and MHC class II positive cells in theblood of these patients are extremely low compared to those found in theblood of B-CLL patients.

Two brothers both with X-linked infantile hypogammaglobulinemia who wereB cell deficient showed different immunophenotypic changes in therelative number of CD3⁺ cells on treatment of their blood. The youngerbrother who was 2 months old and not ill, on treatment of his blood,showed a slight increase in the relative number of CD3⁺ cells which wasaccompanied by a decrease in the relative number of CD3⁻CD19⁻ cells. Onthe other hand, the other brother who was 2 years old and was extremelysick and with a relatively high number of activated T cells expressingthe DR antigens showed a decrease in the number of CD3⁺ cells ontreatment of his blood. No other markers were used to measure otherimmunophentypic changes that might have occurred because the bloodsamples obtained from these two patients were extremely small (Table 2,ID 43/BD and 04/BD).

Patient 91 in Table 2 shows a decrease in the relative number of CD3⁺cells following treatment of blood which was accompanied by an increasein the relative number of CD3⁻CD19⁻ cells. However, on analysis of othersurface markers such as CD4 and CD8 (see Table 3) the patient wasobserved to have a high relative number of CD4⁺CD8⁺ cells in his bloodand this was noted prior to treatment of blood samples with monoclonalantibody to the β-chain of the DR antigen and these double positivecells decreased appreciably following treatment of blood. Furthermore,when further markers were analyzed the relative number of CD3⁺ cellswere seen to have elevated (See Table 4).

An enriched preparation of B-lymphocytes obtained from healthy blooddonors when treated with monoclonal antibody to the β-chain of DRantigens showed a dramatic increase in the relative number of CD3⁺ cellswhich were always accompanied by a decrease in the relative number ofCD19⁺ cells and by an increase in the relative number of CD19⁻CD3⁻cells. Further analysis using markers such as CD4 and CD8 show aconcomitant increase in the relative number of these markers. However,an enriched preparation of T lymphocytes of the same blood donors whentreated with the same monoclonal antibody did not show the same changes.

CD4 and CD8 Panel

The CD4 antigen is the receptor for the human immunodificiency virus.The CD4 molecule binds MHC class II antigen in the B2 domain, a regionwhich is similar to the CD8 binding sites on class I antigens. Bindingof CD4 to class II antigen enhances T cell responsiveness to antigensand so does the binding of CD8 to class I antigens. The CD8 antigens arepresent on the human supressor/cytotoxic T-lymphocytes subset as well ason a subset of natural killer (NK) lymphocytes and a majority of normalthymocytes. The CD4 and CD8 antigens are coexpressed on thymocytes andthese cells lose either markers as they mature into T-lymphocytes.

On analysis of the CD4 and CD8 markers—see below—and from a majority ofblood samples presented in Table 2, a pattern of staining emerges whichsupports the presence of a retrodifferentiation process of B-lymphocytesinto undifferentiated cells and the subsequent differentiation intoT-lymphocytes.

CD4⁺CD8⁺ cells, which are double positive cells, always appearedfollowing treatment of blood samples with monoclonal antibody to thehomologous region of the β-chain and these types of cells were markedlyincreased in the blood of treated samples of patients with B-CLL andwhich were absent altogether in untreated samples (See Table 3 andCharts 1, 2, 3 & 4). In the same specimens the relative number of singlepositive cells such as CD8⁺ and CD4⁺ cells was also noted to increasesimultaneously. Furthermore, a decrease in the relative number ofCD4⁻CD8⁻ cells which, at least in the case of B-CLL correspond to Bcells was noted to fall dramatically in treated samples when compared tountreated specimens which remained at the same level when measured withtime. However, measurement of the relative number of CD4⁺CD8⁺ cells withtime in treated samples showed that there was a concomitant increase inthe number of single positive cells with a decrease in the relativenumber of double positive cells. This type of immunophenotypic change ischaracteristic of thymic development of progenitor cells of theT-lymphocyte lineage in the thymus (Patient number 2, 3 and 4). The CD4antigen is present on the helper/inducer T-lymphocyte subsets (CD4⁺CD3⁺)and a majority of normal thymocytes. However, this antigen is present inlow density on the cell surface of monocytes and in the cytoplasm ofmonocytes and macrophages (CD3⁻CD4⁺).

The relative number of CD4⁺ low cells was affected differently indifferent blood samples following treatment. The relative number of thistype of cells seems unaffected in blood samples of patients with B-CLLfollowing treatment when compared to untreated samples. Such low levelsof CD4 expression is found on monocytes and very early thymocytes.

Patient HIV⁺25 on treatment showed a substantial increase in the numberof double positive cells expressing CD4 and CD8 simultaneously. On theother hand, patient 91 on treatment showed a decrease in this subtype ofcells and the observation of such phenomenon is time dependent. Therelative number of CD8⁺ cells was observed to increase in untreatedblood samples of patients with B-CLL when measured with time whereas therelative number of CD4⁺ and CD4⁺ low cells was observed to decrease atthe same times (Table 3 patient 2, 3 and 4).

DR and CD3 Panel

The DR markers are present on monocytes, dendritic cells, B-cells andactivated T-lymphocytes.

Treated and untreated samples analysed with this panel showed similarimmunophenotypic changes to those obtained when blood samples wereanalysed with the CD19 and CD3 markers (see Table 2) and these antigensas mentioned earlier are pan B and T-cell markers respectively.

Treatment of blood with monoclonal antibodies seems to affect therelative number of DR⁺ B-lymphocytes so that the level of DR+ cellsdecrease. In contrast, the relative number of CD3⁺ (T-cells) cellsincrease significantly (see Table 4 and Chart). Furthermore, therelative number of activated T cells increased in the majority oftreated blood samples of patients with B-CLL and these types of cellswere affected variably in treated samples of patients with otherconditions. Furthermore, the relative number of DR high positive cellsappeared in significant numbers in treated samples of patients withB-CLL and a 6 day old baby with increased DR⁺CD34⁺ blasts in his blood.However, it should be noted that the blasts which were present in thispatient's blood were negative for T and B-cell markers before and aftertreatment but became more positive for myeloid lineage antigensfollowing treatment. The relative number of CD3⁻DR⁻ cells increased inthe majority of treated blood samples and was proportional to increasesin the relative number of CD3⁺ cells (T-cells) and was inverselyproportional to decreases in the relative number of DR+ cells (B-cells).

CD56&16 and CD3 Panel

The CD56&CD16 markers are found on a heterogeneous group of cells, asubset of lymphocytes known generally as large granular lymphocytes andnatural killer (NK) lymphocytes. The CD16 antigen is expressed onvirtually all resting NK lymphocytes and is weakly expressed on someCD3⁺ T lymphocytes from certain individuals. This antigen is found ongranulocytes in lower amount and is associated with lymphocytescontaining large azurophilic granules. The CD16 antigen is the IgG FCreceptor III.

A variable number of CD16⁺ lymphocytes coexpress either the CD57 antigenor low-density CD8 antigen or both. In most individuals, there isvirtually no overlap with other T-lymphocyte antigens such as the CD5,CD4, or CD3 antigens. The CD56 antigen is present on essentially allresting and activated CD16⁺ NK lymphocytes and these subsets of cellscarry out non-major histocompatibility complex restricted cytotoxicity.

Immunophenotyping of treated and untreated blood samples of B-CLL andsome other patients with other conditions showed an increase in therelative number of cells coexpressing the CD56&CD16 antigens which wereheavily granulated and of medium size (see Table 5 and Charts 1, 2, 3 &4). These observations were also accompanied by a marked increase in therelative number of cells expressing the CD3 antigen only (without theexpression of CD56 and CD16 markers) and cells coexpressing theCD56&CD16 and CD3 markers together.

In Table 5, patient numbers 2, 3, and 4 represent the same blood samplebut being analysed at 2 hours, 6 hours and 24 hours respectively (beforeand after treatment). This sample shows that treatment of blood withmonoclonal antibody to the homologous region of the β-chain of DRantigen seems to cause spontaneous production of CD56⁺ and CD16⁺ cells,CD3⁺ cells and CD56⁺ and CD16⁺ CD3⁺ cells and these observations werealways accompanied by the disappearance of B-cell markers (CD19, DR,CD56, CD16⁻CD3⁻).

Onward analysis of this blood sample before and after treatment showedthe levels of CD56⁺ and CD16⁺ cells to decrease with time and the levelof CD3⁺ cells to increase with time.

Blood samples of patient 7 with B-CLL, did not show any changes in thenumber of cells expressing the CD56, CD16 and CD3 antigens when comparedto immunophenotypic changes observed in treated and untreated samplesand this is because the amount of monoclonal antibody added wasextremely low relative to the number of B lymphocytes. However,treatment of this patient's blood sample on a separate occasion with anappropriate amount of monoclonal antibody showed significant increasesin the relative number of CD3⁺, CD56⁺& CD16⁺ and CD56⁺ and CD16⁺CD3⁺cells.

Blood samples of other patients with other conditions showed variablechanges in the level of these cells and this seems to be dependent onthe number of B-lymphocytes present in blood before treatment, durationof treatment and probably the clinical condition of patients.

CD45 and CD14 Panel

The CD45 antigen is present on all human leukocytes, includinglymphocytes, monocytes, polymorphonuclear cells, eosinophils, andbasophils in peripheral blood, thymus, spleen, and tonsil, and leukocyteprogenitors in bone marrow.

The CD14 is present on 70% to 93% of normal peripheral blood monocytes,77% to 90% of pleural or peritoneal fluid phagocytes. This antigen isweakly expressed on granulocytes and does not exist on unstimulatedlymphocytes, mitogen-activated T lymphocytes, erythrocytes, orplatelets.

The CD45 antigen represents a family of protein tyrosine phosphatasesand this molecule interacts with external stimuli (antigens) and effectssignal transduction via the Scr-family members leading to the regulationof cell growth and differentiation.

Engagement of the β-chain of the DR antigens in treated blood samplesespecially those obtained from patients with B-CLL suggests that such atreatment affects the level of CD45 antigens on B-lymphocytes. Theoverall immunophenotypic changes that took place on stimulation of theβ-chain of the DR antigen seem to give rise to different types of cellsthat can be segregated on the basis of the level of CD45 and CD14expression as well as morphology as determined by forward scatter andside scatter (size and granulity respectively) and these results arepresented in Table 6 and Charts (1, 2, 3 & 4). See also FIG. 5 whichdemonstrates the appearance of CD45⁻CD14⁻ cells after treatment with theCR3/43 antibody. These cells are not haempoietic cells.

On treatment the relative number of CD45 low cells (when compared tountreated samples) increased significantly and so did the relativenumber of cells co-expressing the CD45 and CD14 antigens. This type ofimmunophenotypic changes coincided with a decrease in the relativenumber of CD45 high cells (compared to untreated samples). However, thislatter population of cells can be further divided on the basis ofmorphology and the degree of CD45 expression. One type was extremelylarge and had extremely high levels of CD45 antigen when compared to therest of cells present in the charts (see charts 1, 2, 3 and 4). Onanalysis of this panel following treatment with time (see Table 6patient 2, 3 and 4 and chart 1) the relative number of CD45⁺ cellsinitially fell drastically with time to give rise to CD45 low cells.However, analysis of blood 24 hours later showed the opposite situation.

Samples 5 and 7 reveal opposite immunophenotypic changes to thoseobtained with other samples obtained from other B-CLL patients and thisis because the samples were analysed at a much earlier incubation timewith the monoclonal antibody. In fact the sequential analysis of bloodsamples after treatment seems to suggest that the immunophenotypicchanges undertaken by B lymphocytes is time dependent because itrepresents a stage of development and the immunophenotypic changesmeasured at time X is not going to be the same at time X plus (its notfixed once induced). However, these types of changes must be occurringin a more stringent manner in the body otherwise immunopathology wouldensue. The effect of treatment of blood samples from other patients withno B-cell malignancy show variable changes in immunophenotypes of cellsand this because B-lymphocytes are present in lower amount. However,treatment of enriched fractions of B-lymphocytes obtained from healthyblood donors show similar immunophenotypic changes to those obtainedwith B-CLL with high B lymphocyte counts.

CD8 and CD3 Panel

The CD8 antigenic determinant interacts with class I MHC molecules,resulting in increased adhesion between the CD8+ T lymphocytes and thetarget cells. This type of interaction enhances the activation ofresting lymphocytes. The CD8 antigen is coupled to a protein tyrosinekinase (p56ick) and in turn the CD8/p56ick complex may play a role inT-lymphocyte activation.

Treatment of blood samples obtained from patients with B-CLL withmonoclonal antibody to the B chain causes a significant increase in therelative number of CD3CD8 and CD3 (highly likely to be CD4CD3) positivecells thus indicating more clearly that double positive cells generatedinitially are undergoing development into mature T-lymphocytes. This isa process that can be measured directly by CD19 and by DR and indirectlyby CD8⁻CD3⁻ antigens. Serial assessment of treated blood samples of thesame patient with time seems to agree with a process which is identicalto thymocyte development (Table 7, patient 2, 3 and 4 and Chart 1).

The relative number of CD8⁺ cells increased with time in treated anduntreated samples but to a higher extent in untreated samples. On theother hand, the relative number of CD8⁺CD3⁺ cells decreased with time inuntreated samples. However, the relative number of CD3⁺ cells increasedin treated blood samples when measured with time and these types ofcells highly correspond to CD4⁺CD3⁺ single positive cells; a maturerform of thymocytes. In addition, since these samples were alsoimmunophenotyped with other panels (mentioned above in Tables 3, 4, 5and 6) the overall changes extremely incriminate B cells in thegeneration of T lymphocyte progenitors and progenies.

Blood samples from a patient with B-CLL (number 2, 3 and 4 Tables 1, 2,3, 4, 5, 6, 7) in separate aliquots were treated with nothing, PEconjugated monoclonal antibody to the homologous region of the β-chainof DR antigen and unconjugated form of the same monoclonal antibody. Oncomparison of PE conjugated treatment clearly indicates no change in therelative number of CD3 positive cells and associated markers such as CD4which have been observed in significant levels when the same bloodsample was treated with unconjugated form of the antibody. However, anincrease in the number of CD45 positive cells with no DR antigen beingexpressed on their surface was noted when measured with time (see Table8). A finding that was similar to that noted in untreated samples whenimmunophenotyped with time (Table 6). Furthermore, the relative numberof cells expressing CD45 low decreased in time, a phenomenon which wasalso noted in the untreated samples (when measured with time) of thesame patient (see chart 1A).

C. Comparison of the Effect of Other Monoclonal Antibodies withDifferent Specificity on T-Lymphophoiesis

CD19 and CD3 Panel

Treatment of blood samples with monoclonal antibody to the homologousregion of the α-chain of the DR antigen and the homologous region of MHCClass I antigens decreased the number of CD3⁺ cells and increased thenumber of CD19⁺ cells. Treatment of the same blood with monoclonalantibody to the homologous region of the β-chain of the DR antigendecreased the number of CD19⁺ cells and increased the number of CD3⁺cells. Treatment with the latter monoclonal antibody withcyclophosphoamide revealed the same effect (Table 14 patient 5/6 withB-CLL at 2 hr treatment).

Onward analysis of CD19⁺ and CD3⁺ cells in the same samples revealedfurther increases in the relative number of CD3⁺ cells only in bloodtreated with monoclonal antibody to the homologous region of the β-chainof DR antigen (Table 14 patient 5/6 at 24 hours following treatment).However, onward analysis (24 hours later patient 5/6 Table 14) of bloodsamples treated with cyclophosphamide plus monoclonal antibody to theβ-chain of DR antigen show reversal in the relative number of CD19⁺ andCD3⁺ cells when compared to that observed at 2 hour incubation timeunder exactly the same condition.

In general, treatment of blood samples of the same patient withmonoclonal antibody to the homologous region of the α chain of the DRantigen or monoclonal antibody to the homologous of the α-chain of theclass I antigen shows an increase in the relative number of CD19⁺ cells(pan B marker) when compared to untreated sample. The relative number ofCD19⁻CD3⁻ cells decreased slightly in blood samples treated withmonoclonal antibody to the α-chain of DR antigen or treated withmonoclonal antibody to class I antigens (see Table 14 & Charts 2, 3 &4). Treatment of blood samples of patient 09 with monoclonal antibody toclass I antigens increased the relative number of CD3⁺ cells anddecreased slightly the relative number of CD19⁺ and CD19⁻CD3⁻ cells.However, treatment of an enriched preparation of B-lymphocytes obtainedfrom healthy blood donors with monoclonal antibody to the β-chain orα-chain of DR antigen showed similar immunophenotypic changes to thoseobtained with patient with B-CLL.

Treatment of HIV⁺ and IgA deficient patients with monoclonal antibody tothe β-chain of the DR antigen increased the relative number of CD3⁺cells and decreased the relative number of CD19⁺ cells. However,treatment of the same blood sample with monoclonal antibody to thehomologous region of class I antigen did not produce the same effect.Treatment of blood samples obtained from patients (34/BD and 04/BD) withB-cell deficiency showed variable immunophenotypic changes when treatedwith monoclonal antibodies to the β-chain of the DR antigen, class Iantigens and CD4 antigen.

CD4 and CD8 Panel

Blood samples analysed using the CD19 and CD3 panel (Table 14) were alsoimmunophenotyped with the CD4 and CD8 panel (Table 15). Both panels seemto agree and confirm each other. Incubation for 2 hours of blood samplesof patients with B-CLL (Table 15, patients 5/6 and 10, Charts 2, 3 & 4)with monoclonal antibody to the homologous region of the β-chain of theDR antigen or with this monoclonal antibody plus cyclophosphoamideincreased the relative number of CD8⁺ and CD4⁺ cells and cellscoexpressing both markers. On the other hand, treatment of the samesamples with monoclonal antibodies to the homologous region of theα-chain of the DR antigen or the homologous region of the α-chain ofclass I antigen did not produce the same effects.

Comparison of immunophenotypic trends obtained at 2 hours and 24 hoursincubation periods with monoclonal antibody to the β-chain of the DRantigen plus cyclophosphoamide revealed reverse changes in the relativenumber of CD4 and CD8 positive cells (Table 15, patient 5/6 with B-CLLat 2 hours and 24 hours) and such changes were in accordance with thoseobtained when the same blood sample was analysed with the CD19 and CD3panel (Table 14 the same patient). The later findings indicate that thesubsequent differentiation is reversible as the undifferentiated cellscan differentiate into T-lymphocytes or B-lymphocytes.

DR and CD3 Panel

The immunophenotypic changes obtained with DR and CD3 (Table 16) panelconfirm the findings obtained with CD19 and CD3 panel and CD4 and CD8panel (Tables 14 & 15 & Charts 2, 3 & 4) which followed treatment of thesame blood samples with monoclonal antibodies to the homologous regionof the beta- or alpha-side of the DR antigen or monoclonal antibody toclass I antigens or monoclonal antibody to the β-chain of the DR antigenplus cyclophosphoamide at 2 hour analysis.

From the results, it would appear that the monoclonal antibody to thehomologous region of the β-chain of the DR antigen is extremely capableof driving the production of CD3 positive cells from DR⁺ cells.

Furthermore, treatments such as those involving engagement of theα-chain of DR antigens or engagement of the β-side of the molecule inconjunction with cyclophosphoamide (prolonged incubation time) promotedincreases in the relative number of CD19⁺ cells or DR⁺ cells.

CD56&16 and CD3 Panel

Treatment of blood samples, especially of those of patients with B-CLLwith high B-lymphocyte counts with monoclonal antibody to the homologousregion of the β-chain of the DR antigen increased the relative number ofCD56&16 positive cells.

In these patients the relative number of CD3⁺ and CD56⁺ and CD16⁺CD3⁺cells also increased following treatment of blood samples withmonoclonal antibody to the β-chain, confirming earlier observationsnoted with the same treatment when the same blood samples were analysedwith CD3 and CD19 and DR and CD3 panels.

CD45 and CD14 Panel

Blood samples treated with monoclonal antibodies to the β- oralpha-chains of the DR antigen or to the β-chain plus cyclophosphoamideor class I antigens were also analysed with the CD45 and CD14 panel(Table 18). The delineation of CD45 low, CD45 high and CD45 medium isarbitrary. Treatment of blood sample 5/6 (at 2 hours) with monoclonalantibodies to the β-chain of the DR antigen or with this monoclonalantibody plus cyclophosphoamide generated CD45⁺ low cells and increasedthe relative number of CD45⁺ medium cells. However, the former treatmentincreased the relative number of CD45⁺ high cells and the lattertreatment decreased the relative number of CD45⁺ medium cells and thesechanges appeared to be time dependent.

Blood samples of patient 5/6 and 10 (B-CLL) on treatment with monoclonalantibody to class I antigens showed a decrease in the relative number ofCD45⁺ medium cells and similar observations were noted in blood samples09 and HIV⁺ following the same treatment when compared to untreatedsamples. Treatment of blood samples of HIV+ and IgA/D patients withmonoclonal antibody to class I antigen increased the relative number ofCD45⁺ low cells when compared to untreated samples or samples treatedwith monoclonal antibody to the β-chain of the DR antigen. However,blood samples of these patients showed a decrease in the relative numberof CD45⁺ medium cells on treatment with monoclonal antibody to thehomologous regions of the β-chain of the DR antigen. Medium CD45⁺ cellsincreased in blood samples of IgA/D patient following monoclonalantibody to class I antigen treatment. Cells that were extremely large,heavily granular and expressing intense levels of CD45 antigen werenoted in treated blood samples with monoclonal antibody to thehomologous region of the β-chain of DR antigen of MHC class II antigens(see Charts 1, 2, 3 & 4).

CD8 and CD28 Panel

The CD28 antigen is present on approximately 60% to 80% of peripheralblood T (CD3⁺) lymphocytes, 50% of CD8⁺ T lymphocytes and 5% of immatureCD3-thymocytes. During thymocyte maturation, CD28 antigen expressionincreases from low density on most CD4⁺CD8⁺ immature thymocytes to ahigher density on virtually all mature CD3⁺, CD4⁺ or CD8⁺ thymocytes.Cell activation further augments CD28 antigen density. Expression of theCD28 also divides the CD8⁺ lymphocytes into two functional groups.CD8⁺CD28⁺ lymphocytes mediate alloantigen-specific cytotoxicity, that ismajor histocompatibility complex (MHC) class I-restricted. Suppressionof cell proliferation is mediated by the CD8⁺CD28⁻ subset. The CD28antigen is a cell adhesion molecule and functions as a ligand for theB7/BB-1 antigen which is present on activated B lymphocytes.

Treatment of blood samples of patients (Table 19, patients 5/6 and 8)with B-CLL with monoclonal antibody to the homologous region of β-chainof the DR antigen increased the relative number of CD8⁺, CD28⁺ andCD8⁺CD28⁺ cells and all other types of treatments did not.

CD34 and CD2 Panel

The CD34 antigen is present on immature haematopoietic precursor cellsand all haematopoietic colony-forming cells in bone marrow, includingunipotent (CFU-GM, BFU-E) and pluripotent progenitors (CFU-GEMM, CFU-Mixand CFU-blast). The CD34 is also expressed on stromal cell precursors.Terminal deoxynucleotidyl transferase (TdT)⁺B- and T-lymphoid precursorsin normal bone are CD34⁺, The CD34 antigen is present on early myeloidcells that express the CD33 antigen but lack the CD14 and CD15 antigensand on early erythroid cells that express the CD71 antigen and dimlyexpress the CD45 antigen. The CD34 antigen is also found on capillaryendothelial cells and approximately 1% of human thymocytes. Normalperipheral blood lymphocytes, monocytes, granulocytes and platelets donot express the CD34 antigen. CD34 antigen density is highest on earlyhaematopoietic progenitor cells and decreases as the cells mature. Theantigen is absent on fully differentiated haematopoietic cells.

Uncommitted CD34⁺ progenitor cells are CD38⁻, DR⁻ and lacklineage-specific antigens, such as CD71, CD33, CD10, and CD5, whileCD34+ cells that are lineage-committed express the CD38 antigen in highdensity.

Most CD34⁺ cells reciprocally express either the CD45RO or CD45RAantigens. Approximately 60% of acute B-lymphoid leukaemia's and acutemyeloid leukaemia express the CD34 antigen. The antigen is not expressedon chronic lymphoid leukaemia (B or T lineage) or lymphomas. The CD2antigen is present on T lymphocytes and a subset of natural killerlymphocytes (NK).

The results are shown in Charts 2, 3 and 4.

Analysis of blood samples of a patient with B-CLL (Table 20, patient 5/6at 2 hours) after treatment with monoclonal antibodies to the β-chain ofthe DR antigen or the α-chain of the same antigen revealed markedincreases in the relative number of CD34⁺ and CD34⁺CD2⁺ cells aftertreatment with the former antibody. Since the same blood samples wereimmunophenotyped with the above mentioned panels (see Tables 14 to 19)for other markers the increase in the relative number of CD34⁺ andCD34⁺CD2⁺ cells observed here seems to coincide with increases in therelative number of CD4⁺CD8⁺, CD8⁺CD3⁺ and CD4⁺CD3⁺ single positive (SP)cells. Furthermore, these findings which seem exclusive to engagement ofthe β-chain of the HLA-DR antigen, are in direct support that theprocess is giving rise to T-lymphopoiesis via B lymphocyte regression.

On analysing the same treatment 24 hours later the CD34⁺ cells seemed todecrease in levels to give rise to further increase in the relativenumber of T lymphocytes. The process of retrodifferentiation thatinitially gave rise to T-lymphopoiesis can be reversed to give rise toB-lymphopoiesis. The former phenomenon was observed at 2 hoursincubation time with monoclonal antibody to the β-chain of the HLA-DRantigen plus cylophosphoamide, whereas the latter process was noted at24 hours incubation time with the same treatment in the same sample(Chart 2).

Treatment of blood samples of HIV⁺ patient (Table 20 patient HIV+) withmonoclonal antibody to the β-chain of the HLA-DR antigen markedlyincreased the relative number of CD34⁺ and CD2⁺CD34⁺ cells and so didtreatment of the same blood sample with monoclonal antibody to theβ-chain of the HLA-DR antigen and monoclonal antibody to the α-chain ofthe same antigen when added together. However, treatment of this bloodsample with monoclonal antibody to the α-chain of the HLA-DR antigen didnot affect the level of CD34⁺ cells. Treatment of blood samples obtainedfrom a 6-day old baby (BB/ST Table 20) who was investigated at that timefor leukaemia and who had very high number of atypical cells (blasts) inhis blood with monoclonal antibody to the β-chain of the HLA-DR antigen,or monoclonal antibody to the α-chain of the same antigen or bothmonoclonal antibodies added together resulted in the followingimmunophenotypic changes.

On analysis of untreated blood samples the relative number of CD34⁺ andDR⁺ cells were markedly increased and on treatment with monoclonalantibody to the β-chain the relative number of CD34⁺ cells furtherincreased but were noted to decrease on treatment with monoclonalantibody to the α-chain of the HLA-DR antigen or treatment withmonoclonal antibodies to the α and β-chains of the molecule when addedtogether. However, the latter treatment increased the relative number ofCD34⁺CD2⁺ cells and the opposite occurred when the same blood sample wastreated with monoclonal antibody to the β-chain of the HLA-DR antigenalone. On analysis of treated and untreated blood aliquots of the samepatient 24 hours later the relative number of CD34+ decreased with allabove mentioned treatments except it was maintained at a much higherlevel with monoclonal antibody to the β-chain of the HLA-DR antigentreatment. The latter treatment continued to decrease the relativenumber of CD34⁺CD2⁺ cells 24 hours later.

These results indicate that engagement of the HLA-DR antigen via theβ-chain promotes the production of more CD34⁺ cells from CD2⁺CD34⁺ poolor from more mature types of cells such as B-lymphocytes of patientswith B-CLL and these results indicate that this type of treatmentpromotes retrodifferentiation. However, immunophenotyping of bloodsamples 24 hours later suggests that these types of cells seem to existin another lineage altogether and in this case cells seem to exist orrather commit themselves to the myeloid lineage which was observed onanalysis of treated blood sample with the CD7 and CD13&33 panel.

Morphology changes immunophenotypic characteristics of B-lymphocytes ofB-CLL and enriched fractions of healthy individuals (using CD19 beads)on treatment with monoclonal antibodies to homologous regions of theβ-chain of MHC class II antigens. These were accompanied by a change inthe morphology of B-lymphocytes. B-lymphocytes were observed colonisingglass slides in untreated blood smears were substituted by granulocytes,monocytes, large numbers of primitive looking cells and nucleated redblood cells. No mitotic figures or significant cell death were observedin treated or untreated blood smears.

The results of Table 20 also demonstrate a further important finding inthat according to the method of the present invention it is possible toprepare an undifferentiated cell by the retrodifferentiation of a moremature undifferentiated cell.

D. Microscope Pictures

In addition to the antigen testing as mentioned above, the method of thepresent invention was followed visually using a microscope.

In this regard, FIG. 6 is a microscope picture of differentiated B cellsbefore the method of the present invention. FIG. 7 is a microscopepicture of undifferentiated cells formed by the retrodifferentiation ofthe B cells in accordance with the present invention wherein the agentwas a monoclonal antibody to the homologous regions of the β-chain ofHLA-DR antigen. The undifferentiated cells are the dark stained clumpsof cells. FIG. 8 is a microscope picture of the same undifferentiatedcells but at a lower magnification.

FIGS. 6 to 8 therefore visually demonstrate the retrodifferentiation ofB cells to undifferentiated stem cells by the method of the presentinvention.

FIG. 9 is a microscope picture of differentiated B cells before themethod of the present invention. FIG. 10 is a microscope picture ofundifferentiated cells formed by the retrodifferentiation of the B cellsin accordance with the present invention wherein the agent used was amonoclonal antibody to the homologous regions of the β-chain of HLA-DRantigen. Again, the undifferentiated cells are the dark stained clumpsof cells. FIG. 11 is a microscope picture of the formation ofdifferentiated granulocyte cells from the same undifferentiated cells ofFIG. 10.

FIGS. 9 to 11 therefore visually demonstrate the retrodifferentiation ofB cells to undifferentiated stem cells by the method of the presentinvention followed by commitment of the undifferentiated cells to newdifferentiated cells being of a different lineage as the originaldifferentiated cells.

These microscopy experiments have also been performed with blood fromBCLL patients, treated with the CR3/43 monoclonal antibody as describedabove. As discussed above, blood from BCLL cells is a useful aid instudying the retrodifferentiation process because the blood containshigher than normal numbers of B lymphocytes. The results are shown indetail in FIGS. 12 to 15.

FIG. 12 shows at two different magnifications, an untreated blood samplefrom a BCLL patient. The untreated B lymphocytes (blue cells) showtypical morphology, i.e. condensed chromatin structure and sparsecytoplasm. The remaining cells are erythrocytes (red blood cells).

Treatment of blood samples with antibody CR3/43 leads initially toclustering of B lymphocytes into aggregates (FIG. 13).

The clustered B cells gradually lose their typical morphology,characterised by the formation of cobblestone-like-cell areas,decondensation of chromatin structure, appearance of prominent nucleoli,enlargement of cell volume and cytoplasmic basophilia typical ofundifferentiated cells (FIG. 14). Relaxed (decondensed) chromatinstructure is an important feature of undifferentiated cells as comparedto differentiated cells. This is likely to be due to a need for moreextensive access to transcriptional units to determine changes in geneexpression required for commitment along a given cell lineage. Bycontrast, it is well known that more differentiated cell have a morecondensed chromatin structure since only a small amount of chromatinneeds to be transcriptionally active.

The appearance of undifferentiated cells is always accompanied by theappearance of cells (15A to 15J) with differentiated morphology.Importantly, these cells could not have arisen by proliferation, since(i) the incubation time was too short for one or more complete celldivisions to take place (ii) no mitotic figures are seen and (iii) theabsolute number of leucocytes remained the same before and aftertreatment. Furthermore less differentiated progenitors were seen inassociation with their more differentiated progenies (see the myeloidprecursor in FIG. 15J), indicating that these specialized cells arose bydifferentiation.

Micrographs FIG. 15A to 15J show the types of differentiated cells seenfollowing treatment of B-CLL lymphocytes with CR3/43 monoclonalantibodies: Platelets (P1)—FIG. 15A, Neutrophils (Ne)—FIG. 15B,Eosinophils (Eso)—FIG. 15C, Megakaryocytes (Meg)—FIG. 15D, Basophils(Ba)—FIG. 15G, Lymphocytes (Ly)—FIG. 15H, Monocytes (Mo)—FIG. 15I andMyeloid progenitors (Mp)—FIG. 15J. Also seen were erythroid progenitorsand macrophages (data not shown).

Thus, in summary, these microscopy results show changes in B cellmorphology in samples from BCLL patients, who have high levels of matureB lymphocytes. The microscopy pictures show changes in the morphology ofthe B lymphocytes, which initially cluster, followed by the appearanceof various cells with a graded range of morphologies from progenitorcells to differentiated cells (neutrophils, basophils, eosinophils,megakaryocytes, platelets, lymphocytes, macrophages, granulocytes, stabgranulocytes and stromal-like cells).

In addition, and very importantly, the presence of erythroid and myeloidprogenitors is seen (FIG. 15J—and data not shown). The myeloidprogenitor is clearly distinguishable morphologically from the othercells, being larger and with a distinct nuclear morphology as well ascontaining cytoplasmic granules.

The microscopy data therefore support morphologically what the flowcytometry data indicate in terms of cell surface markers. These dataallow one to conclude that treatment of B lymphocytes with an antibodyto MHC HLA-DR β chain results in a decrease in the numbers of Blymphocytes and an increase in the number of cells of other haemapoieticlineages including immature precursor cells.

The retrodifferentiation of T cells treated with an antibody to an MHCclass II α-chain (monoclonal antibody TAL.1B5) to undifferentiated stemcells by the method of the present invention followed by commitment ofthe undifferentiated cells to new differentiated cells being of adifferent lineage as the original differentiated cells was also followedby microscopy (data not shown).

E. Analysis of VDJ Recombination Rearrangements in RetrodifferentiatedLymphocytes

By way of background, the differentiated cells used in these experiments(B lymphocytes or cells with certain properties of T lymphocytes) havegenes which have already undergone rearrangement to encode a mature Igor a TCR, respectively. In the process or rearranging, intermediateportions of DNA that are not part of the final, expressed TCR or Iggene, primarily DNA which is between the variable (V) region encodingsegment and the constant (C) region-encoding segment of these receptors,are spliced out of the genome. These excised fragments are retained inthe cell in the form of extrachromosomal DNA. For the cells to trulyretrodifferentiate, the excised DNA would be reinserted into the genome,placing the cells in a state similar to that preceding their originaldifferentiation. Because of this, a probe complementary to a sequence inthe rearranged gene will be expected to hybridize to a larger DNArestriction fragment when the DNA has returned to its unrearranged orgerm line state as compared to the rearranged DNA that characterizes thedifferentiated state.

1. Rearrangement of TCR Genes in Daudi Cells

In the experiment resulting in the Southern blot shown in FIG. 16, awell-known cell line, Daudi, a B-cell lymphoma with one rearranged TCRgene (and the other deleted), was used. Genomic DNA was prepared fromDaudi cells and digested with EcoRI, subjected to gel electrophoresisand probed with a labeled TCR β-chain DNA probe. Daudi cells were usedrather than B lymphocytes purified from human patients because thesecells are clonally related and form a homogenous cell population withthe same gene rearrangements that can be clearly viewed by Southernblotting of digested genomic DNA. In a normal blood sample, differentcells have different rearrangements and so a Southern blot would appearas a smear.

A functional gene encoding the TCR β-chain is assembled in lymphocytesby a series of somatic rearrangements that occur during lymphocytematuration to bring together a V segment, a D segment and a J segment. Avery clear explanation of these rearrangement processes is given inGenes VI, Lewin, Oxford University Press, 1997 (pages 1994-1023)—astandard undergraduate textbook. Particular pages are cited below.

Firstly, a D segment is joined by a recombination process to one ofseveral J segments in a D-J joining reaction. Then, one of the manypossible V segments (<60) is joined to the resulting DJ segment (V-Djoining) to form a complete TCR β-chain gene. The constant region geneis immediately downstream of the rearranged VDJ segment, although theremay be intervening J segments which are spliced out during RNAprocessing to bring the constant gene exon into proximity with therearranged VDJ gene segment (Lewin, p 998).

In human cells, there are two different TCR β-chain constant region genesegments, denoted Cβ1 and Cβ2, present at two different loci, each ofwhich is preceded by a cluster of six or seven joining region (Jβ) genesegments (Jβ1 and Jβ2) and one D segment (Dβ1 and Dβ2) (see FIG. 1,Toyonaga et al., 1985, Proc. Natl. Acad. Sci. USA 82: 8624-8628 andLewin, p 1017).

The recombination events which lead to the V, D and J-C segments beingbrought into proximity are catalysed by a multitude of proteins,including RAG-1 and RAG-2 which recognise nonamer and heptamer sequencespresent at the recombining ends of the V, D and J-C gene sequences.Depending on the orientation of these nonamer/heptamer sequences,recombination results either in an inversion or a deletion. Both typesof events will result in a change in the restriction enzyme fragmentpattern of the genomic DNA. Furthermore, a deletion event does notnecessarily result in complete loss of the excised fragment. Rather, theends of the excised fragment are rejoined to produce a circle of DNAwhich remains in the cell (Okazaki et al., 1987, Cell 49: 477-85; Daviset al., 1991, J. Exp. Med. 173: 743-6; Livak and Schatz, 1996, Mol. CellBiol. 16: 609-18; Harriman et al. 1993, Annu Rev Immunol. 11:361-84).Each gene segment, of course, has two alleles since cells have a diploidchromosome complement.

In the normal germline state, the Cβ1 and Cβ2 genes are arranged asshown in FIG. 1, Toyonaga et al., 1985. A restriction digest of genomicDNA with EcoRI will generate two relevant bands detectable by the probeused in the experiment (the probe is a labelled DNA fragment derivedfrom Cβ1 which also hybridises to Cβ2 due to a high degree of sequencehomology): (i) a 12 kb band containing Cβ1 sequence; and (ii) a 4 kbband containing Cβ2 sequence. This germline configuration is seen inundifferentiated immature cells (lane A of FIG. 16) This germlineconfiguration is also perfectly illustrated by lane 3 (2 hours withCR3/43 antibody) of FIG. 16, giving an identical pattern to that of laneA.

In the differentiated state, both alleles of Cβ1 and Cβ2 genes arerearranged such that there is no longer a 12 kb fragment at the Cβ1locus or a 4 kb fragment at the Cβ2 locus. In fact, no hybridisingfragment derived from the Cβ1 locus is present on the gel (this is dueto deletion of the hybridising sequence from both Cβ1 alleles as aresult of recombination). As for the Cβ2 locus, there are actually nowtwo major bands corresponding to different “alleles” resulting fromrearrangements on both chromosomes. The largest band, which is smallerthan 4 kb, corresponds to a fragment of one of the two rearrangedalleles. The lowest band is a fragment of the other rearranged allele.The intermediate minor band is probably derived from a subclone of Daudicells with a different rearrangement—hence its presence in a submolaramount to either allele. Nonetheless, the rearranged state is veryclearly shown in lane 1 where both major bands are clearly visible.

2 hours with the negative control antibody (TAL.1B5) which binds to theα-chain of MHC-DR actually results in the loss of the upper band,whereas the lowest band has a similar intensity to the untreated cellsin lane 1 (see lane 2). A possible explanation for this is that thecells are differentiating, further resulting in a further recombinationevent at the Cβ2 locus of one allele, which leads to loss of Cβ2sequences. This is entirely consistent with known phenomena.

24 hours with the negative control antibody appears to restore the threebands seen in the untreated cells (see lane 4). However the bandsactually migrate at a lower position than the bands seen in lane 2. Itis not quite clear how this has arisen. A possible explanation is thatreintegration of deleted sequences has occurred, consistent with thelooping-out-excision-reintegration model (Malissen et al., 1986, Nature319: 28-32). Nonetheless, neither result seen with the TAL.1B5 antibodyat 2 hours or 24 hours is indicative of a rearrangement to the germlinepattern. Lanes 2 and 4 actually represent a negative control—theantibody to the α-chain does not result in restoration of the germlinesequences.

By contrast, the results obtained with a monoclonal antibody (CR3/43) tothe β-chain of MHC-DR after two hours show a pattern of bands thatcorrespond to the germline configuration, namely a 12 kb band and a 4 kbband (compare lane 3 with lane A). In other words, these results showthat the germline restriction pattern at the Cβ1 and Cβ2 loci has beenrestored for all alleles.

From these results we conclude that the pattern of bands seen in lane 3are indicative of a rearrangement of the genomic DNA of thedifferentiated cells to regenerate the germline configuration.

The importance of this finding should not be understated. Never beforehas it been demonstrated that a genomic rearrangement, includingdeletions, can be reversed to restore the genome to the state in whichit existed before the differentiation process took place. The mostlikely explanation is that the inversion caused by the rearrangement ofthe Cβ2 alleles during differentiation has been reversed, and thedeletion of the Cβ1 sequence that caused loss of the 12 kb bands hasalso been reversed. The source of the missing Cβ1 sequence is likely tobe episomal circular DNA present in the nucleus from the originaldeletion event. The existence of this circular DNA has been cataloguedin the prior art (see references cited above). Nonetheless, the precisemechanism by which this restoration of the germline genome has occurredis not important. What is important is that it has occurred.

A continued incubation with the monoclonal antibody (CR3/43) to theβ-chain of MHC-DR for 24 hours results in a more complex banding pattern(lane 5). However these bands do not represent the same bands as in theuntreated control. In particular, fragments of about 12 kb thathybridize to the probe are still present (“Cβ2 alleles”). Further, it isimportant to appreciate that the bands marked “Cβ2 alleles” do notcorrespond to the smaller than 4 kb band seen in the untreated control(lane 1). The most likely explanation for the results seen in lane 5 isthat a secondary rearrangement process has occurred since thehybridization pattern resembles that of T-cells in that it ischaracterized by a rearranged TCR gene (this explanation is consistentwith the flow cytometry data showing an increase in cells having cellmarkers characteristic of T cells). Nonetheless, regardless of theprecise molecular explanation, the results seen in lane 5 at 24 hoursexposure to the CR3/43 antibody are supportive of the results obtainedat 2 hours exposure in lane 3.

2. Rearrangement of Ig Gene in B-CLL Cells

The Southern blot shown in FIG. 17B was obtained using peripheral bloodcells from patients with chronic lymphocytic leukemia (B-CLL). GenomicDNA was prepared from these largely monoclonal B cells and digested withBamHI and HindIII, subjected to gel electrophoresis and probed with alabeled TCR DNA probe. These B-CLL cells were treated for 24 hours withthe CR3/43 (anti class II MHC chain of HLA-DR, DP and DQ) which wasdescribed above. The blots were probed with a radiolabeled Ig J regionprobe. The two bands obtained from the untreated cells in lane A,represent the two rearranged Ig alleles (paternal and maternal). Thesebands did not appear in lane B which shows the pattern 24 hours afterantibody treatment of cells. In their place appeared a 5.4 kb bandcharacteristic of the germ line Ig gene.

In another experiment, shown in FIG. 17A, cells were left untreated ortreated for the times indicated with the anti-class II MHC β-chainantibody. The Ig VDJ region was amplified by PCR in the differentiated(control) and antibody-treated B-CLL cells (left half of gel). Thisgenerated a VDJ amplification product from the untreated cells. However,no such band was observed in the antibody-treated cells because, as aresult of insertion of the excised genomic DNA, this “germ line” DNAconfiguration was not susceptible to PCR amplification using theparticular primers for VDJ. A similar experiment (right side of gel)allowed me to visualize the behavior of a control, housekeeping, geneencoding β-actin. There was no difference in the β-actin PCRamplification product, regardless of treatment. Thus, this “control”gene did not appear to be affected by the retrodifferentiation processthat caused profound alterations in the Ig gene of the same cells underthe same conditions.

The results presented above show that treatment of cells with an agentthat engages an appropriate cell surface receptor inducesretrodifferentiation of these cells that is proven at the molecularlevel (and monitored) by observing the retrogression of therearrangements of chromosomal DNA that characterize the differentiatedstate. Thus, it is concluded on the basis of the molecular genetic andmorphological evidence that cells of the B lymphocyte lineage, treatedwith an agent (mAb) that engages the class II MHC β-chain, undergoretrodifferentiation. By contrast, the same cells treated withantibodies that engage class II α-chain are not similarly induced toretrodifferentiate. If anything, they appear to differentiate (forward)along the B cell pathway.

F. Further Studies on Retrodifferentiation of B Lymphocytes

FACsVantage puried BCLL cells (95% pure B cells) from BCLL patients weretreated with the CR3/43 antibody as described above and the cellsprocessed by flow cytometry. The results shown below in Table A confirmfurther the results obtained above. A significant increase in the numberof CD34⁺ cells was obtained together with a large reduction in thenumber of cells having cell surface markers characteristic of the Blymphocyte lineage (CD19, CD20 and CD22). An important point to notefrom Table A is that it also shows an increase in the number of cellsthat are both CD34 negative and lineage negative. These undifferentiatedcells are not committed to the haemopoietic lineage and precede CD34⁺stem cells in differentiation. Further, examination of samples by lightmicroscopy showed a range of adherent cell types having morphologicalcharacteristics of non-haemopoietic cells.

TABLE A Marker 0 HR 2 HR 24 HR CD20 73 67 16 CD14 0 3 23 CD34 0 1 23 CD70 2 0 CD16 8 3 2 CD19 95 71 1 CD22 5 3 2 CD33 0 0 0 CD3 0 0 0

The loss of CD19 cell surface markers accompanied by the appearing ofCD34 cell surface markers on the same cell has also been demonstratedand recorded on video in real time using confocal microscopy.B-lymphocytes before the addition of CR3/43 mab stained green with aFITC conjugated monoclonal antibody to CD19. After the addition ofCR3/43 mab, cells lost their green fluorescence and began to stain redwith a PE/Cy5 (or quantum red) conjugated monoclonal antibody to CD34but not green (see FIG. 21 which shows two still images from thetimelapse video). The results clearly confirm that during B lymphocyteretrodifferentiation, lineage specific markers such as CD19 are lostwhilst a stem cell marker such as CD34 is re-expressed.

G. Other Agents that Induce Retrodifferentiation of B Lymphocytes toHaemopoietic Stem Cells

Initial studies actually identified threeagents—granulocyte/monocyte-colony stimulating factor (GM-CSF),erythropoietin and mAb CR3/43. A preparation of enriched, purified,normal B lymphocytes was treated with one of these three agents in asimilar manner to that described for CR3/43 and TAL.1B5 above andtreated samples examined by flow cytometry as described above. Comparedwith the negative control, all three samples treated with either GM-CSF,erythropoietin or mAb CR3/43 showed changes consistent withretrodifferentiation. In particular, all three agents increased therelative number of CD34⁺ cells in the cell population (see FIG. 18). Thegreatest effect, however, was seen with CR3/43 and consequently, thisagent was selected for use in the more detailed studies presentedherein.

H. Properties of Haemopoietic Stem Cells Produced by theRetrodifferentiation Process

Colony Forming Assays

To confirm that the CD34⁺ cells observed by flow cytometry and theundifferentiated cells identified by microscopy had the properties ofundifferentiated haemopoietic cells, blood samples treated with anantibody to the class II WIC β-chain (CR3/43—see above) were subjectedto colony forming assays—a standard method known in the art forassessing the capabilities of primitive haemopoietic cells.

In vitro clonal assays for hematopoietic stem cell allows thequantification of primitive progenitor cells that possess the ability toproliferate, differentiate and develop into phenotypically andfunctionally mature myeloid and/or erythroid cells. For example in thepresence of growth factors stem cell when seeded/immobilised in soft-gelmatrix in vitro are capable of clonal growth (proliferation) anddifferentiation.

FIG. 19 is a colony assay of stem cells produced according to themethods of the invention, using inverted bright-field microscopy. Inthis assay B cells obtained from buffy coat of healthy blood donors weretreated with CR3/43 mab and then subjected to colony assays as describedin the materials and methods section.

Panels (a) to (e) in FIG. 19 show:

-   a) Bright field microscopy of culture dish viewed at ×3    magnification showing erythroid, myeloid and mixed (consisting of    mature myeloid and erythroid cells) colonies which can be seen    readily even by the naked eye. Each colony arose from a single    haematopoietic stem cell by proliferation and subsequent    differentiation.-   b) MIX-CFC this colony arose from a single multi-potent    haematopoietic stem cell (stem cells capable of giving rise to cells    of myeloid and erythroid lineages.-   c) M-CFC this colony consists of macrophages.-   d) GM-CFC this colony consist of the myeloid lineage including    macrophages, granulocyte and megakaryocytes-   e) BFU-E this colony consists of cells belonging to the erythroid    lineage such as normoblasts and non-nucleated red cells. The red    colouration of cells shows that they are well hemogloblinized. The    large size of this colony indicates that it arose from an extremely    primitive stem cell.

The same results were obtained with B-CLL cells (data not shown).Untreated B cells did not give rise to haematopoietic colonies (data notshown). These results therefore demonstrate the presence of viablehaemopoietic stem cells in blood samples treated with monoclonalantibody CR3/43 to the class II MHC β-chain but not in untreated bloodsamples.

Long Term Culture

The long-term assay examines the self-renewal potential ofhaematopoietic stem cells. In this culture most components of bonemarrow haematopoiesis are reproduced in vitro. The important feature ofthis culture is sustained haematopoiesis, which occurs in the absence ofadded growth factors. In this assay the process of hematopoiesis isabsolutely dependent upon the establishment of an adherent layer of bonemarrow derived stromal cells. Stromal cells (consisting of a variety ofnon-haemopoietic cells e.g., fibroblast, fat cells and including allcell types belonging to the mesenchymal system) support haematopoiesisby providing the appropriate environment (secretion of growth factorsand synthesis of extracellular matrix) to promote the survival,self-renewal, proliferation and differentiation of the stem cells.

In this assay, treatment of B cells obtained from buffy coats of healthyblood donors (the same results were obtained with B-CLL cells) withCR3/43 mab gave rise to the formation of an adherent cell layer withinhours of adding the antibody which, also increased with time.

The adherent layer consisted of stromal cells (blanket cells, consistingmainly of fibroblast/mesenchymal-type cells/light refringent large cellswhen viewed with inverted bright field microscopy—see FIG. 20) whichsupported the growth and development of haematopoietic cells up to 12weeks and longer (these cells show intimate contact with haematopoieticcells). Also visible in the adherent layer are groups of primitivehaematopoietic cells (also known as cobblestone areas/clusters of darkappearing cells) which are the origin of prolonged production ofhaematopoietic cells.

The non-adherent layers which are on top of the stromal layer (clustersof bright appearing cells) consisting of small round cells formingclusters of haematopoietic foci. This layer contains stem cells and alsomore committed progenitors of the haematopoietic system. The nonadherent layer was capable of giving rise to MIX-CFC, GM-CFC, M-CFC,BFU-E (as determined using the clonal assay) and CFU-F (colony formingunit-fibroblast) (when sub-cultured with long term culture medium).

I. RT-PCR of Cells Treated with an Antibody to the β-Chain of HLA-DR.

Gene transcription was measured in Ramos (B lymphoma) and K562(erythroid leukaemia) cells treated with the CR3/43 mab for the CD34,c-kit (ligand of stem cell factor), ε-haemoglobin (embryonic form ofhaemoglobin) and β-actin genes.

Methods

mRNA was extracted before and after treatment with CR3/43 mab usingRNAZOL (CINA BIOTECH). mRNA were subjected to hexamer priming reversetranscription by incubating at room temperature for 5 mins with 4 μlstandard buffer, 2 μl dNTPs, 1 μl RNASIN, 1 μl reverse primer (randomhexamer primer) and 1 μl MMLV reverse transcriptase enzyme. This mixturewas further incubated for 1 hr at 38° C. Mixtures were then subjected toPCR under standard conditions using primers designed to amplify CD34,ε-kit, ε-haemoglobin and β-actin sequences. Primers were synthesised atthe Randell Institute Kings College according to published data.

Results

The results obtained show that whereas the levels of β-actin mRNA didnot change, the levels of CD34, c-kit and ε-haemoglobin mRNA allincreased significantly following treatment with the CR3/43 mAb. Theresults for CD34 and c-kit provide further support for the data detailedabove that demonstrate the retrodifferentiation of B lymphocytes toproduce haemopoetic stem cells.

The results obtained for the ε-haemoglobin are even more interestingsince ε-haemoglobin is normally only expressed in embryonic cells. It istherefore possible that treatment with the CR3/43 mAb not only givesrise to haemopoietic stem cells but also to even more primitiveundifferentiated cells such as embryonic stem cells.

J. Summary

In short, the examples describe in vitro experiments that revealextremely interesting, seminal findings regarding the ontogeny anddevelopment of T and B lymphocytes which can be utilised in thegeneration of stem cells to affect lymphohaematopoiesis in peripheralblood samples in a matter of hours.

Treatment of peripheral blood samples obtained from patients with B-cellchronic lymphocytic leukaemia's (B-CLL) with high B lymphocyte counts,with monoclonal antibody to the homologous region of the β-chain ofclass-II antigens gave rise to a marked increase in the relative numberof single positive (SP) T lymphocytes and their progenitors which weredouble positive for the thymocyte markers CD4 and CD8 antigens and thesewere coexpressed simultaneously. However, these phenomena were alwaysaccompanied by a significant decrease in the relative number ofB-lymphocytes. These observations were not noted when the same bloodsamples were treated with monoclonal antibodies to the homologous regionof the α-chain of class-II antigens or to the homologous region ofclass-I antigens.

Treatment of whole blood obtained from patients with B-cell chroniclymphocytic leukaemia (CLL) with monoclonal antibody to the homologousregion of the B chain of the HLA-DR antigen appeared to give rise toT-lymphopoiesis. This event was marked by the appearance of doublepositive cells coexpressing the CD4 and CD8 markers, the appearance ofcells expressing CD34 and the concomitant increase in the number ofsingle positive CD4⁺CD3⁺ and CD8⁺CD3⁺ lymphocytes. Furthermore, theimmunophenotypic changes that took place in the generation of such cellswere identical to those cited for thymocyte development, especially whenmeasured with time.

The percentages of double positive cells (DP) generated at 2 hourincubation time of whole blood with monoclonal antibody to thehomologous region of the β-chain of the DR antigen, decreased with timeand these events were accompanied by increase in the percentages ofsingle positive CD4⁺CD3⁺ and CD8⁺CD3 cells simultaneously and at latertimes too. TCR α and β chains were also expressed on these types ofcells.

B-lymphocytes were constantly observed to lose markers such as CD19,CD21, CD23, IgM and DR and this coincided with the appearance of CD34⁺and CD34⁺CD2⁺ cells, increases in CD7⁺ cells, increases in CD8⁺CD28⁺ andCD28⁺ cells, increases in CD25⁺ cells, the appearance of CD10⁺ and CD34⁺cells and CD34⁺ and CD19⁺ cells increases in CD5⁺ cells, and cellsexpressing low levels of CD45 antigen. These changes were due totreatment of blood with monoclonal antibody to the homologous region ofthe β-chain of HLA-DR antigen.

The immunophenotypic changes associated with such treatment isconsistent with retrodifferentiation and subsequent commitment (i.e.recommitment) of B lymphocytes, because the majority of white bloodcells in blood of patients with B-CLL before treatment were Blymphocytes. Furthermore, B-lymphocytes of patients with B-CLL whichwere induced to become T-lymphocytes following treatment withcyclophosphamide and monoclonal antibody to the β-chain of HLA-DRantigen, were able to revert back to B lymphocytes following prolongedincubation with this treatment.

On analysis of treated samples with monoclonal antibody to the β-chainof HLA-DR antigen, with CD16&56 and CD3 and CD8 and CD3 panels, therelative number of cells expressing these markers steadily increases inincrements consistent with those determined with panels such as CD19 andCD3 and DR and CD3. Investigation of the supernatant of treated anduntreated samples of patients with HIV infection using nephlometry andimmunoelectrophoresis reveals increased levels of IgG indicating thatthe B-cells must have passed through the plasma cell stage. The increasein the relative number of all above-mentioned cells was also accompaniedby the appearance of medium size heavily granulated cells expressing theCD56&16 antigens in extremely high amounts. Other cells which wereextremely large and heavily granulated were observed transiently andthese were positive for CD34 and double positive for CD4 CD8 markers.Other transient cells were also observed and these were large andgranular and positive for the CD3 and CD19 receptors. CD25 which waspresent on the majority of B-lymphocytes was lost and became expressedby newly formed T-lymphocytes which were always observed to increase innumber.

CD28⁺CD8⁺ and CD28⁺ cells appeared after treatment of whole blood ofpatients with B-CLL with monoclonal antibody to the homologous region ofthe B chain of the DR antigen. These findings were due to treatment ofblood with monoclonal antibody to the homologous region of the β-chainof HLA-DR antigen.

T-lymphopoiesis generated in this manner was also observed in peripheralblood of healthy blood donors, cord blood, bone marrow, patients withvarious infections including HIV⁺ individuals and AIDS patients,enriched fractions for B lymphocytes obtained from blood samples ofhealthy blood donors, IgA deficient patients and other patients withvarious other conditions. Furthermore, analysis of myeloid markers intreated samples of two patients with B-CLL with monoclonal antibody tothe homologous region of the β-chain of the HLA-DR antigen showed asignificant increase in the relative number of cells expressing themyeloid markers such as CD13 and CD33. These markers were coexpressedwith the CD56 & 16 or the CD7 antigens. However, the relative number ofCD7⁺ cells with T-lymphocyte markers and without myeloid antigens wasobserved on a separate population of cells. These particularobservations were not seen in untreated samples or in samples treatedwith monoclonal antibodies to class I antigens or the homologous regionof the α-chain of HLA-DR antigen (see Charts 2 & 3). These final resultssuggest that B-lymphocytes once triggered via the β-chain of the HLA-DRantigen are not only able to regress into T lymphocyte progenitor cellsbut are also capable of existing into the myeloid and erythroidlineages.

Thus in summary, the data presented in the present applicationdemonstrate that (i) it is possible to convert healthy cells from onelineage to cells having the cell surface markers and morphologicalcharacteristics of cells of several other lineages and (ii) it ispossible to obtain cells having the cell surface markers andmorphological characteristics of primitive precursor cells (for examplestem cells), from differentiated B lymphocytes and T lymphocytes.

It should be noted that a number of experiments have been carried outwith BCLL cells. BCLL cells are mature B lymphocytes that are incapableof differentiating to the final terminally differentiated stage of aplasma cell. Instead, due to a chromosome defect, they exhibit highlevels of proliferation, hence the large numbers of B lymphocytes in theblood of BCLL patients. By contrast to a number of tumour cellsdescribed in the prior art, BCLL cells have not undergone any form oflimited reverse differentiation prior to use in the methods of theinvention. Furthermore they do not exhibit any characteristics ofundifferentiated cells in term of genomic structure, cell markers orcell morphology. They are in all respects mature B lymphocytes.

Thus whereas some malignant cells may have to a limited extent somecharacteristics of undifferentiated cells, this is not the case for BCLLcells, which are a perfectly acceptable experimental system for studyingB lymphocytes. In fact BCLL and Daudi cells are not sufficientlydistinguished from normal cells in any aspects relevant to theseexperiments. Indeed, the suitability of BCLL cells as a model system isconfirmed by Martensson et al., 1989, Eur. J. Immunol. 19: 1625-1629(see page 1625 rhs, 1^(st) para).

It should be noted that the stem cells that are produced by the methodof the present invention may be stem cells of any tissue and are notnecessarily limited to lymphohaematopoietic progenitor cells.

Other modifications of the present invention will be apparent to thoseskilled in the art.

TABLE 1 CLINICAL DIAGNOSIS OF PATIENTS AND EXPERIMENTAL CONDITIONS OFBLOOD SAMPLES INCLUDING COULTER COUNTS (WBC) FOLLOWING AND PRIORTREATMENT OF BLOOD SPECIMENS WITH VARIOUS MONOCLONAL ANTIBODIES ANDOTHER AGENTS WBC/L # LYMPH/L PATIENT EXPT X10-9 % LYMPH 10X-9 AGENT IDDIAGNOSIS COND B A B A B A ML/mL 1 B-CLL 12 HR 100 ND 86.1 ND 86.1 NDANTI-B AT 22 C. 50 2 B-CLL 2 HR 39.1 9.6 74.4 63.3 29.9 6.1 ANTI-B AT 22C. 50 2 HR AT 39.1 37.7 74.4 75.1 29.9 28.3 ANTI-B 22 C. PE 50 3 B-CLL 6HR 39.5 9.3 71.9 67.2 28.3 6.2 ANTI-B AT 22 C. 50 6 HR 39.5 37.7 71.972.5 28.3 27.4 ANTI-B AT 22 C. PE 50 4 B-CLL 24 HR 39 9.3 73 66.5 28.46.2 ANTI-B AT 22 C. 50 24 HR 39 36.2 73 70.4 28.4 25.5 ANTI-B AT 22 C.PE 50 5 B-CLL 2 HR ANTI-B AT 22 C. 50 ANTI-A 50 ANTI-I 50 ANTI-B & TOXICAGENT 25 + 25 6 B-CLL 24 HR ANTI-B AT 22 C. 50 7 B-CLL 24 HR 170 12895.4 91.1 16.9 11.6 ANTI-B AT 22 C. 178 94.2 16.8 10 130 90.4 11.9ANTI-I 10 ANTI-B & TOXIC AGENT 10 + 20 8 B-CLL 24 HR 16 7 81.9 51.2 143.0 ANTI-B AT 22 C. 20 9 B-CLL 12 HR +++ 89.5 87 85.1 +++ 76.2 ANTI-B AT22 C. 85.4 30 +++ +++ ANTI-I 89.4 30 +++ 84.9 +++ ANTI-4 95.4 30ANTI-I+II+4 10 + 10 + 10 10 B-CLL 2 HR 19.3 ND 86 ND 16.7 ND ANTI-B AT22 C. 30 ANTI-I 30 92 OUT 2 HR 5.4 ND 74.5 ND ND ANTI-B PATIENT AT 22 C.20 87 OUT 2 HR 4.8 ND 59.3 ND ND ANTI-B PATIENT AT 22 C. 20 91 OUT 2 HR4.2 ND 54.0 ND ND ANTI-B PATIENT AT 22 C. 20 21 OUT 2 HR 3.9 ND 47.4 NDND ANTI-B PATIENT AT 22 C. 20 34 OUT 2 HR 7.2 ND 20.0 ND ND ANTI-BPATIENT AT 22 C. 20 36 CMV 4 HR 13.4 ND 7.3 ND ND ANTI-B INFANT AT 22 C.20 93 HIV+ 4 HR 5.6 ND 43.4 ND ND ANTI-B INFANT AT 22 C. 20 BB/ST 40%BLAST 2 HR AT 60.5 ND 20.2 ND 12.2 ND ANTI-B IN BLOOD 22 C. 50 6 DAYSOLD 24 HR ANTI-A AT 22 C. 50 ANTI-AB 25 + 25 HIV25 AIDS 2 HR 7.5 ND 34.8ND 2.6 ND ANTI-B AT 22 C. 50 ANTI-A 50 ANTI-AB 25 + 25 43/BD B CELL 4 HRANTI-B DEFICIENT AT 22 C. 20 ANTI-I 20 ANTI-4 20 0B/BD B CELL 4 HRANTI-B DEFICIENT AT 22 C. 20 ANTI-I 20 ANTI-4 20 HIV+ AIDS 6 HR ANTI-BAT 22 C. 20 ANTI-I IgA-D IgA 6 HR ANTI-B DEFICIENT AT 22 C. 20 ANTI-I 20EXPT COND: EXPERIMENTAL CONDITIONS B: BEFORE A: AFTER ANTI-B: monoclonalantibody to the homologous region of the β-chain of HLA-DR anigenANTI-A: monoclonal antibody to the homologous region of the α-chain ofHLA-DR antigen ANTI-I: monoclonal antibody to the homologous region ofClass I antigens ANTI-AB: both ANTI-B and ANTI-A added togather ANTI-4:monoclonal antibody to the CD4 antigen ANTI-I+II+4: ANTI-I and ANTI-Band ANTI-4 added togather Cytoxic agent: Cyclophophamide ML/ml: microlitre per ml L: litre

TABLE 2 IMMUNOPHENOTYPING OF PATIENTS WITH B-CLL AND OTHER CONDITIONSBEFORE AND AFTER TREATMENT OF BLOOD SAMPLES WITH MONOCLONAL ANTIBODY TOTHE HOMOLOGOUS REGION OF THE B CHAIN OF THE HLA-DR WITH CD19 AND CD3MONOCLONAL ANTIBODIES. % CD19+HGC % CD19+ % CD3+ % CD19+CD3+ % CD3−CD19−D3− FC+ PATIENT B A B A B A B A B A 1 88 40 5 19 1 2 6 26 0 12 2 73 1510 33 2 7 15 41 0 5 3 73 11 11 33 2 2 14 52 0 2 4 71 13 11 37 2 2 16 470 2 5 85 40 5 16 1 1 6 26 3 18 6 85 43 5 18 1 1 6 27 3 10 7 90 72 2 4 02 7 8 0 14 8 62 25 7 13 0 1 29 55 2 6 9 90 85 2 3 0 0 2 1 1 4 10 78 50 714 0 0 14 26 0 8 92 12 10 38 49 0 1 49 40 0 0 91 7 3 35 29 0 1 59 67 0 087 5 3 32 38 1 1 63 58 0 0 21 1 1 27 29 1 0 71 70 0 0 34 1 1 13 13 0 286 84 0 0 39 10 6 23 25 0 0 67 69 0 0 93 6 3 26 27 1 1 68 70 0 0 BB/ST 11 12 13 0 0 87 86 0 0 HIV25 7 2 26 27 0 0 68 67 0 0 43/BD 0 0 40 42 0 158 54 0 0 04/BD 0 0 49 41 0 3 43 41 0 0 HIV+ 1 1 10 14 0 0 89 87 0 0IgA/D 10 1 21 25 2 3 67 71 0 0 B: before treatment. A: after treatment

TABLE 3 IMMUNOPHENOTYPING OF PATIENTS WITH B-CLL AND OTHER CONDITIONSBEFORE AND AFTER TREATMENT OF BLOOD SAMPLES WITH MONOCLONAL ANTIBODY TOTHE B CHAIN OF THE HOMOLOGOUS REGION OF THE HLA-DR WITH MONOCLONALANTIBODIES TO CD4 AND CD8. % CD8+ % CD4+ % CD4+CD8+ % CD4−CD8− CD4+LOWPATIENT B A B A B A B A B A 1 2.8 16 2.9 11.4 0 3.2 93.1 67.6 0 0 2 6.213.2 9.1 24.3 0 9.4 78.7 46 5.8 6.3 3 7.2 13.1 7.4 23.9 0 8.2 78.8 48.16.3 6.6 4 10.1 24.2 7.6 24.9 0.3 2.8 77.5 42 4.6 5 5 2.9 16.2 1.8 7.6 02 95 62.3 0 0 6 ND 12 ND 8.1 ND 1.7 ND 75.7 ND 0 7 1.9 2.6 1.9 2.8 0 095.8 94.3 0 0 8 3.2 7 3.9 6.9 0.1 2 87.3 79.8 4.3 6 9 2.8 2.9 3 3 0 0 9494.1 0 0 10 5.7 9.4 4.7 9.1 0.6 0.8 88.7 79.2 0 0 92 21 19 21.6 21 0.81.9 50.5 52.5 5.3 4.8 91 15.4 18.1 13.6 17.9 6.2 2.6 57 57.3 7.3 3.5 8716.8 21.8 13.4 20.4 2.9 2.6 59.5 48.9 7 5.6 21 16 24.1 9.1 15.2 1 2.669.6 53.2 3.7 4.2 34 9.4 11.9 5.7 4.9 2 3.3 67.6 65.3 14.4 14.5 39 12.112.6 13.1 14.6 0.4 1.3 62.3 66.7 11.9 4.3 93 18.9 20.3 9.7 10.3 1.8 1.465.5 65.9 3.4 1.8 BB/ST 6.3 13 5.7 7.3 2.2 1.1 34.7 70.3 50.3 7.6 HIV2524.1 24.9 0.8 1.1 1.3 5 70.2 69.3 2.9 3.8

TABLE 4 IMMUNOPHENOTYPING OF PATIENTS WITH B-CLL AND OTHER CONDITIONSBEFORE AND AFTER TREATMENT OF SAMPLES WITH MONOCLONAL ANTIBODY TO THE BCHAIN OF THE HLA-DR WITH MONOCLONAL ANTIBODIES TO CD3 AND DR DR+ CD+CD+DR+ DR−CD3− DR+HCD3− PATIENT B A B A B A B A B A 1 87 45.5 3.5 20.82.5 4.2 6.9 21.6 0 7.6 2 76.2 19.4 9.6 29.2 3.9 8.7 10.3 36.8 0 5.5 377.7 18.3 8.4 29.4 4.1 8.8 9.6 38.1 0 4.7 4 76.8 19.2 7.6 29.5 6.2 10.59.1 37.2 0 3.3 5 ND 47.1 ND 11.5 ND 9.9 ND 22.4 ND 7.3 6 ND 7 91.4 85.82.4 2.5 0.7 0.7 5.1 4.2 0 6.3 8 61.8 28.9 6.5 11.2 2 3.3 28.6 54.6 0 1.59 ND 10 82.6 44.7 4.3 9.8 3.3 5 9.8 22.2 0 17.9 92 23.8 14.1 39.3 41.94.5 3.5 32.4 40.5 0 0 91 13.3 7.9 29.6 32.5 3.4 2.9 53.4 56.5 0 0 8714.8 12.2 284 34.1 5.5 6.6 51.1 46.5 0 0 21 ND 34 11.9 12.9 10.4 13.70.8 0.6 76.7 72.8 0 0 39 25.6 13.7 24.6 25.2 3 2.8 46.5 25.2 0 0 93 13.38.9 18.4 18.9 9.9 10.1 58.2 61.7 0 0 BB/ST 44.2 32.5 11.7 12.2 0.8 0.843 49.4 0 4.6

TABLE 5 IMMUNOPHENOTYPING OF PATIENTS WITH B-CLL AND OTHER CONDITIONSBEFORE AND AFTER TREATMENT OF BLOOD SAMPLES WITH MONOCLONAL ANTIBODY TOTHE HOMOLOGOUS REGION OF THE B CHAIN OF THE HLA-DR WITH MONOCLONALANTIBODIES TO CD16+56 AND CD3. CD56+&16 CD3+ CD56+&16+CD3+ CD56+&16−CD3−PATIENTS B A B A B A B A 1 2 4.3 5.7 19.7 0.7 1.7 91.3 73 2 11.5 38.912.4 32.6 1 6.6 74.5 21 3 12 36.2 12.1 34.5 0.7 6 75.5 23 4 12.2 32.612.4 39.6 0.5 5 74.7 22.2 5 ND 13.1 ND 9.4 ND 2.6 ND 73.5 6 ND 7 0.8 0.82,8 2.4 0.3 0.2 96.2 96.4 8 24.8 52 5.4 12.4 0.9 4.1 68.3 31.1 9 ND 101.1 1.3 6.1 13.7 2.1 2.5 90.5 82.4 92 23.8 34.5 44.3 44.8 2 1.5 29.218.6 91 4.6 3.9 28.8 29.4 3 3.2 63.3 63.3 87 47.9 46.4 28.8 36.5 5.8 3.716.9 13 21 9.4 9.4 19.7 23.6 4.2 6.7 66 59.5 34 21.5 12.8 11.4 13.7 1.80.6 64.6 72.8 39 7 21 23.4 26.1 1.1 0.1 68.2 71 93 55.8 54.9 26.2 26.31.7 2 16.1 16.8 BB/ST 28.8 29.9 12 14.3 0.8 1.8 49.4 53.6

TABLE 6 IMMUNOPHENTYPING OF PATIENTS WITH B-CLL AND OTHER CONDITIONSBEFORE AND AFTER OF TREATMENT OF BLOOD WITH MONOCLONAL ANTIBODY TO THEHOMOLOGOUS REGION OF THE B CHAIN OF THE HLA-DR WITH MONOCLONALANTIBODIES TO CD45 AND CD14. CD45+H CD45+L CD45+CD14+ PATIENTS B A B A BA 1 90.5 70.1 7.5 21.9 0.8 3.3 2 85.8 52.2 8.8 38.3 5.3 9.5 3 84.3 52.29.9 33.8 5.1 13.2 4 91.5 79.2 2.1 7 5.7 10.8 5 63.1 84.6 34.9 9.4 0.53.6 6 ND 7 52.8 85.2 45.6 13.9 0.5 0.6 8 71.1 55 71.1 34.5 5.3 8.7 9 SEE10 79.7 47.3 16.3 48 2.1 1.9 92 61.7 64.7 27.4 26.6 5.9 3.6 91 49.4 49.240.4 44.3 6.5 3.2 87 52.4 61.5 36.1 28.7 7 6.5 21 45.8 43.3 44.3 47.66.2 3.3 34 24.4 24.6 54.8 59.6 13.3 9.7 39 48.7 46.3 30.5 42.1 14.5 8.893 SEE HIV+ 22.6 26.9 66.8 63.5 6.8 6.7 IgA/D 47.4 59.8 41.9 33.3 5.94.1

TABLE 7 IMMUNOPHENOTYPING OF PATIENT WITH B-CLL AND OTHER CONDITIONSBEFORE AND AFTER TREATMENT OF BLOOD WITH MONOCLONAL ANTIBODIES TO THEHOMOLOGOUS REGION OF THE B-CHAIN OF THE HLA-DR WITH MONOCLONALANTIBODIES TO CD8 AND CD3. CD8+ CD3+ CD8+CD3+ CD8−CD3− PATIENTS B A B AB A B A 2 0.6 1.3 7.5 19.3 4.2 19.3 87.7 63.8 3 1.1 1.4 8.3 20.3 5.618.4 84.8 59.8 4 3.5 2.9 8.3 27 3.9 16.6 84.2 53.1 92 3.5 1.9 27.6 25.218.4 19 50.3 52.8 91 4 3.1 18.2 19 14.1 12.6 63.6 65.3 87 5.7 3.9 19.923.6 15.4 17.4 58.8 55 21 4.8 7.4 16.3 17.3 13.7 13 65.2 62 34 3 3.6 5.26.7 7.6 7.5 84.1 82.3

TABLE 8 IMMUNOPHENOTYPING OF A PATIENT WITH B-CLL WITH TIME AFTERTREATMENT OF BLOOD WITH PE CONJUGATED MONOCLONAL ANTIBODY TO THEHOMOLOGOUS REGION OF THE B-CHAIN OF THE HLA-DR MEASURE WITH MONOCLONALANTIBODIES TO CD45 AND CD14. TIME DR+CD45+CD14+r CD45+L CD45+H  2 HR81.7 8.2  8.2  6 HR 80.7 8.1 10.6 24 HR 79 1.1 18.4

TABLE 9 IMMUNOPHENOTYPING OF A PATINENT WITH B-CLL WITHTIME AFTERTREATMENT OF BLOOD WITH PE CONJUGATED MONOCLONAL ANTIBODY TO THEHOMOLOGOUS REGION OF THE B-CHAIN OF THE HLA-DR MEASURED WITH MONOCLONALANTIBODIES TO CD19 AND CD3. TIME CD19+DR+r CD3+ CD3+DR+ CD19-CD3-DR-  2HR 87.4 10.1 1.8 10.7  6 HR 75.5 10.4 3.1 10.7 24 HR 74 11.7 2.9 11

TABLE 10 IMMUNOPHENOTYPING OF A PATIENT WITH B-CLL WITH TIME AFTERTREATMENT OF BLOOD WITH PE CONJUGATED MONOCLONAL ANTIBODY TO THEHOMOLOGOUS REGION OF THE B-CHAIN OF THE HLA-DR MEASURED WITH MONOCLONALANTIBODIES TO CD4 AND CD8. CD8+ CD4+&CD8+ CD4+ CD4- TIME &DR+r CD4+&DR+r DR+ CD8-DR-  2 HR 77.6 6.8 5.4 1.3 8.8  6 HR 75.8 6.7 6.4 1.8 9.324 HR 77 6.4 4.8 1.9 11

TABLE 11 IMMUNOPHENOTYPING OF A PATIENT WITH B-CLL WITH TIME AFTERTREATMENT OF BLOOD WITH PE CONJUGATED MONOCLONAL ANTIBODY TO THEHOMOLOGOUS REGION OF THE B-CHAIN OF THE HLA-DR MEASURED WITH MONOCLONALANTIBODIES TO CD3 AND DR. TIME DR+ CD3+ CD3+DR+ CD3+DR-  2 HR 75 9.5 4.210.9  6 HR 74.8 8.8 4.8 10.9 24 HR ND ND ND ND

TABLE 12 IMMUNOPHENOTYPING OF A PATIENT WITH B-CLL WITH TIME AFTERTREATMENT OF BLOOD WITH PE CONJUGATED MONOCLONAL ANTIBODY TO THEHOMOLOGOUS REGION OF THE B-CHAIN OF THE HLA-DR MEASURED WITH MONOCLONALANTIBODIES TO CD16&56 ANDCD3. CD56+ CD56+CD16+ CD56-CD16- TIME 816+DR+rCD3+ &CD3+DR+r &CD16-DR-  2 HR 82.5 9.5 4.1 3.5  6 HR 84.3 7.5 4.1 3.324 HR ND ND ND ND

TABLE 13 IMMUNOPHENOTYPING OF A PATIENT WITHB-CLL WITH TIMEAFTERTREATMENT OF BLOOD WITH PE CONJUGATED MONOCLONAL ANTIBODY TO THEHOMOLOGOUS REGION OF THE B-CHAIN OF THE HLA-DR MEASURED WITH MONOCLONALANTIBODIES TO CD8 AND CD3. CD8+CD+ CD8- TIME CD8+DR+ CD3+ 3&DR+r CD3-DR-2 HR 76.2 6.6 6.7 10.6 6 HR 76.5 6.2 6.2 10.3

TABLE 14 IMMUNOPHENOTYPING OF PATIENTS WITH B-CLL BEFORE AND AFTERTREATMENT OF BLOOD WITH MONOCLONAL ANTIBODIES TO THE HOMOLOGOUS REGIONOF THE A-CHAIN OF THE HLA-DR, THE HOMOLOGOUS REGION OF THE B-CHAIN OFTHE HLA-DR, THE TWO MONOCLONAL TOGETHER, MONOCLONAL TO THE HOMOLOGOUSREGION OF THE B-CHAIN PLUS CYCLOPHOSPHOAMIDE AND THE HOMOLOGOUS REGIONOF CLASS I ANTIGENS MEASURED WITH TIME. CD19+ CD3+ CD19+CD3+ CD19−CD3−ID B A AB A AI B A AB A AI B A AB A AI B A AB A AI A BC A BC A BC A BC5/6 2H 86 91 54 40 89 5 4 16 23 5 1 1 3 2 1 6 4 27 33 5 24 N 88 51 60 86N 4 18 10 4 N 2 1 2 3 N 4 29 28 7 10 2H 77 N 59 N 80 7 N 13 N 7 1 N 1 N0 14 N 26 N 12 09 24 8 N N N 6 32 N N N 38 1 N N N 1 59 N N N 56 43/BD6H 0 N 0 0 0 40 N 42 43 49 0 N 1 0 1 58 N 54 54 47 04/BD 6H 0 N 0 0 0 49N 41 45 46 0 N 3 1 3 43 N 42 44 41 HIV+ 6H 1 N 0 N 1 10 N 14 N 12 0 N 0N 0 89 N 86 N 87 IgA/D 6H 10 N 1 N 12 21 N 25 N 20 2 N 1 N 3 67 N 71 N68 B = Before; A = After; AB = after addition to antibody to beta chain;AA = after addition of antibody to alpha chain; ABC = after addition ofantibody to either alpha or beta chain and cycloposphoamide; AI = afteraddition of antibody to Class I.

TABLE 15 CD8 AND CD4 Error! Bookmark not defined. CD8+ CD4+ CD4+CD8+CD4−CD8− ID B A AB A AI B A AB A AI B A AB A AI B A AB A AI A BC A BC ABC A BC 5/6 2H 3 2 14 10 4 2 2 8 8 3 0 0 3 2 1 95 94 74 79 93 24 N 3 9 44 N 3 8 4 3 N 0 2 2 0 N 94 81 90 93 10 2H 3 N 7 N 4 4 N 7 N 3 1 N 2 N 191 N 83 N 92 09 24 10 N N N 15 21 N N N 38 2 N N N 2 61 N N N 53

TABLE 16 CD3 AND DR DR+ CD3+ CD3+DR+ CD3−DR− ID B A AB A AI B A AB A AIB A AB A AI B A AB A AI A BC A BC A BC A BC 5/6 2H N 90 54 N 87 N 4 12 N4 N 2 10 N 3 N 5 22 N 5 10 2H 83 N 63 N 81 4 N 8 N 4 4 N 7 N 4 9 N 23 N12 09 24 14 N N N 13 30 N N N 36 3 N N N 3 51 N N N 47

TABLE 17 CD16 & 56 AND CD3 CD56+ & 16+ CD3+ CD56+ & 16+CD3+ CD56− &16−CD3− ID B A AB A AI B A AB A AI B A AB A AI B A AB A AI A BC A BC ABC A BC 5/6 2H N 0 13 N 4 N 5 9 N 5 N 1 3 N 1 N 94 74 N 90 10 2H 0 N 1 N1 6 N 14 N 6 1 N 2 N 1 92 N 65 N 92 09 24 42 N N N 41 36 N N N 38 2 N NN 2 20 N N N 19

TABLE 18 CD45 AND CD14 CD45+L CD45+M CD45+H CD45+CD14+ ID B A AB A AI BA A A AI B A AB A AI B A AB A AI A BC A B BC A BC A BC 5/6 2H 0 0 5 10 044 43 50 50 32 55 43 50 31 67 1 1 1 2 0 10 2H 0 N 0 N 0 43 N 54 N 35 54N 42 N 62 1 N 1 N 0 09 24 2 N N N 1 18 N N N 16 71 N N N 76 7 N N N 5HIV+ 6H 4 N 3 N 6 63 N 61 N 41 23 N 27 N 40 7 N 7 N 7 IgA/D 6H 2 N 2 N 440 N 31 N 44 47 N 60 N 44 6 N 4 N 6

TABLE 19 CD8 AND CD28 CD8+ CD28+ CD8+CD28+ CD8−CD28− ID B A AB A AI B AAB A AI B A AB A AI B A AB A AI A BC A BC A BC A BC 5/6 2H N 3 6 N 3 N 14 N 2 N 1 4 N 1 N 95 86 N 94 8 2H 4 N 6 N N 3 N 5 N N 1 N 3 N N 92 N 86N N

TABLE 20 CD34 AND CD2 CD34+ CD2+ CD34+CD2+3 CD34−CD2− ID B A AB A AI B AAB A AI B A AB A AI B A AB A AI A BC A BC A BC A BC 5/6 2H N 1 34 N N N6 13 N N N 3 30 N N N 90 21 N N 24 N 1 6 9 N N 7 23 4 N N 3 33 43 N N 8734 34 N HIV+ 2H 2 1 12 13 N 20 21 21 12 N 4 5 9 14 N 73 73 64 60 N BB/ST2H 26 23 33 14 N 15 14 15 15 N 31 30 23 36 N 27 32 28 35 N 24 N 11 29 11N N 13 12 9 N N 27 9 18 N N 48 49 61 N

CHART 1 IMMUNOPHENOTYPIC CHANGES OF UNTREATED AND TREATED BLOOD SAMPLEOF PATIENT (2, 3 & 4) WITH MONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGIONOF THE β-CHAIN OF HLA-DR ANTIGEN MEASURED WITH TIME. WITHOUT WITH FL1FL2 TIME NOTHING001 WITH002 CD45 CD14  2 HR NO001 WE002 CD45 CD14  6 HR001001 002002 CD45 CD14 24 HR NOTHING003 WITH004 CD3 CD19  2 HR NO003WE004 CD3 CD19  6 HR 001003 002004 CD3 CD19 24 HR NOTHING004 WITH005 CD4CD8  2 HR NO004 WE005 CD4 CD8  6 HR 001004 002005 CD4 CD8 24 HRNOTHING005 WITH006 CD3 DR  2 HR NO005 WE006 CD3 DR  6 HR 001005 002006CD3 DR 24 HR NOTHING006 WITH007 CD3 CD56&16  2 HR N0006 WE007 CD3C056&16  6 HR 001006 002007 CD3 C056&16 24 HR N003 W004 CD3 CD8  2 HRNO007 WE008 CD3 CD8  6 HR 001007 002008 CD3 CD8 24 HR

CHART 1A IMMUNOPHENOTYPIC CHANGES OF UNTREATED AND TREATED BLOOD SAMPLEOF PATIENT (2, 3, 4) WITH MONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGIONOF THE β-CHAIN OF HLA-DR ANTIGEN CONJUGATED TO PE MEASURED WITH TIME. IDFL1 FL2 TIME WL003 CD45 CD14  2 HR WEL003 CD45 CD14  6 HR 003003 CD45CD14 24 HR WL005 CD3 CD19  2 HR WEL005 CD3 CD19  6 HR 003005 CD3 CD19 24HR WL006 CD4 CD8  2 HR WEL006 CD4 CD8  6 HR 003006 CD4 CD8 24 HR WL007CD3 DR  2 HR WEL 007 CD3 DR  6 HR WL008 CD3 CD65&16  2 HR WEL 008 CD3CD56&16  6 HR WL005 CD3 CD8  2 HR WEL009 CD3 CD8  6 HR

CHART 2 IMMUNOPHENOTYPIC CHANGES OF UNTREATED AND TREATED BLOOD OFPATIENT (1) WITH MONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGION OF THEβ-CHAIN OF HLA-DR ANTIGEN, THIS ANTIBODY AND CYCLOPHOSPHAMIDE,MONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGION OF THE α-CHAIN OF KA- DRANTIGEN AND MONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGION OF CLASS IANTIGEN MEASURED WITH TIME. Error! WITH- Bookmark WITH OUT FL1 FL2 TIMENA001 CD45 CD14  2 HR A2B001: AB CD45 CD14  2 HR A2A : AA CD45 CD14  2HR DNAA001: ABC CD45 CD14  2 HR A1001: AI CD45 CD14  2 HR NC001 CD3 CD19 2 HR C2B001: AB CD3 CD19  2 HR C2A001: AA CD3 CD19  2 HR DNAC001: ABCCD3 CD19  2 HR C1001: AI CD3 CD19  2 HR A124H001: AI CD3 CD19 24 HRA2B24H001: AB CD3 CD19 24 HR A2A24H001: AA CD3 CD19 24 HR A2BX24H001: ABCD3 CD19 24 HR ND001 CD4 CD8  2 HR D2B001: AB CD4 CD8  2 HR D2A001: AACD4 CD8  2 HR DNAD001: ABC CD4 CD8  2 HR D1001: AI CD4 CD8  2 HRD124H001: AI CD4 CD8 24 HR D2BX24H001: AB CD4 CD8 24 HR D2B001: AB CD4CD8 24 HR D2A001: AA CD4 CD8 24 HR E1001: AI CD3 DR  2 HR E28001: AB CD3DR  2 HR E2A001: AA CD3 DR  2 HR F1001: AI CD3 CD56&16  2 HR F2B001: ABCD3 CD56&16  2 HR F2A001: AA CD3 CD56&16  2 HR G1001: AI CD28 CD8  2 HRG2A001: AA CD28 CD8  2 HR G2B001: AB CD28 CD8  2 HR H1001: AI CD7CD33&13  2 HR H2A001: AA CD7 CD33&13  2 HR H2B001: AB CD7 CD33&13  2 HRI2A001: AA CD21 CD5  2 HR I2B001: AB CD21 CD5  2 HR J2A001: AA CD34 CD2 2 HR J2B001: AB CD34 CD2  2 HR B2A24H001: AA CD34 CD2 24 HR B2B24H001:AB CD34 CD2 24 HR B2BX24H001: CD34 CD2 24 HR ABC K2B001: AB CD10 CD25  2HR K2A001: AA CD10 CD25  2 HR

CHART 3 IMMUNOPHENOTYPIC CHANGES OF UNTREATED AND TREATED BLOOD OFPATIENT (8) WITH MONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGION OF THEβ-CHAIN OF HLA-DR ANTIGEN. WITH WITHOUT FL1 FL2 TIME AN001 CD45 CD14 2HR A2001 CD45 CD14 2 HR CN001 CD3 CD19 2 HR C2001 CD3 CD19 2 HR DN001CD4 CD8 2 HR D2001 CD4 CD8 2 HR EN001 CD3 DR 2 HR E2001 CD3 DR 2 HRFN001 CD3 CD56&16 2 HR F2001 CD3 CD56&16 2 HR GN001 CD28 CD8 2 HR G2001CD28 CD8 2 HR HN001 CD7 CD5 2 HR H2001 CD7 CD5 2 HR IN001 CD13 CD20 2 HRI2001 CD13 CD20 2 HR JN001 CD45RA CD25 2 HR J2001 CD45RA CD25 2 HR KN001CD57 CD23 2 HR K2001 CD57 CD23 2 HR

CHART 4 IIMMUNOPHENOTYPIC CHANGES OF UNTREATED AND TREATED BLOOD SAMPLEOF PATIENT (10) WITH MONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGION OF THEβ-CHAIN OF HLA-DR ANTIGEN AND MONOCLONAL ANTIBODY TO THE HOMOLOGOUSREGION OF CLASS I ANTIGENS. WITH WITHOUT FL1 FL2 TIME CLL0001 CD45 CD142 HR CLL1001 CD45 CD14 2 HR CLL2001 CD45 CD14 2 HR CLL0003 CD3 CD19 2 HRCD3 CD19 2 HR CLL1003 CD3 CD19 2 HR CLL2003 CD3 CD19 2 HR CLL0004 CD4CD8 2 HR CLL1004 CD4 CD8 2 HR CLL2004 CD4 CD8 2 HR CLL005 CD3 DR 2 HRCLL1005 CD3 DR 2 HR CLL2005 CD3 DR 2 HR CLL0006 CD3 CD56&16 2 HR CLL1006CD3 CD56&16 2 HR CLL2006 CD3 CD56&16 2 HR

I claim:
 1. A method comprising increasing the relative number ofhematopoietic stem cells in a cell population including committed cells,wherein the method comprises contacting in vitro a cell populationderived from unmobilized blood with an antibody that binds to MHC classII antigens, and enriching hematopoietic stem cells or recovering thehematopoietic stem cells from the cell population by using a cellsurface marker, wherein the hematopoietic stem cells are MHC class I⁺and/or MHC class II⁺ cells and wherein the hematopoietic stem cells areenriched or recovered from the cell population 2 hours after the cellpopulation is first contacted with the antibody.
 2. The method accordingto claim 1 wherein the committed cells are noncancer cells.
 3. Themethod according to claim 1 wherein the committed cells aredifferentiated cells.
 4. The method according to claim 3 wherein thecommitted cells are selected from CFC-T cells, CFC-B cells, CFC-Eosincells, CFC-Bas cells, CFC-GM cells, CFC-MEG cells, BFC-E cells, CFC-Ecells, T cells and B cells.
 5. A method according to claim 1 wherein theantibody is a monoclonal antibody.
 6. A method according to claim 5wherein the antibody is selected from the group consisting of monoclonalantibody CR3/43 and monoclonal antibody TAL 1B5.
 7. A method accordingto claim 1, wherein the antibody is used in conjunction with abiological response modifier, wherein the biological response modifieris selected from the group consisting of an alkylating agent, animmunomodulator, a growth factor, a cytokine, and a hormone.
 8. A methodcomprising increasing the relative number of hematopoietic stem cells ina cell population including committed cells, wherein the methodcomprises contacting in vitro a cell population derived from unmobilizedblood with an antibody that binds to MHC class II antigens, inconjunction with cyclophosphamide, wherein the hematopoietic stem cellsare MHC class I⁺ and/or MHC class II⁺ cells.
 9. A method comprisingincreasing the relative number of hematopoietic stem cells in a cellpopulation derived from unmobilized blood, which population includescommitted cells, said hematopoietic stem cells being capable of beingcommitted into more differentiated hematopoietic cells, which methodcomprises: (i) contacting in vivo and initial cell population comprisingthe committed cells with an antibody that binds to MHC class IIantigens, (ii) culturing the cell population thereby producing analtered cell population comprising hematopoietic stem cells, and (iii)enriching said hematopoietic stem cells or recovering said hematopoieticstem cells from the altered cell population, wherein said hematopoieticstem cells are enriched or recovered from the altered cell population 2hours after commencement of the incubation.
 10. The method according toclaim 9 wherein step (iii) comprises enriching said hematopoietic stemcells or recovering said hematopoietic stem cells from the altered cellpopulation by using a cell surface marker.
 11. A method comprisingincreasing the relative number of hematopoietic stem cells in a cellpopulation including committed cells, wherein the method comprisescontacting in vitro a cell population derived from unmobilized bloodwith an agent selected from the group consisting of (a) erythropoietinin combination with an antibody that binds to MHC class II antigens, and(b) GM-CSF in combination with an antibody that binds to MHC class IIantigens, and enriching hematopoietic stem cells or recovering thehematopoietic stem cells from the cell population by using a cellsurface marker, wherein the hematopoietic stem cells are MHC class I⁺and/or MHC class II⁺ cells, and wherein the hematopoietic stem cells areenriched or recovered from the cell population 2 hours after the cellpopulation is first contacted with the agent.
 12. The method accordingto claim 11 wherein the committed cells are noncancer cells.
 13. Themethod according to claim 11 wherein the committed cells aredifferentiated cells.
 14. The method according to claim 13, wherein thecommitted cells are selected from CFC-T cells, CFC-B cells, CFC-Eosincells, CFC-Bas cells, CFC-GM cells, CFC-MEG cells, BFC-E cells, CFC-Ecells, T cells and B cells.
 15. The method according to claim 11 whereinthe antibody is a monoclonal antibody.
 16. The method according to claim15 wherein the antibody is selected from the group consisting ofmonoclonal antibody CR3/43 and monoclonal antibody TAL 1B5.
 17. A methodaccording to claim 11, wherein the agent is used in conjunction with abiological response modifier, wherein the biological response modifieris selected from the group consisting of an alkylating agent, animmunomodulator, a growth factor, a cytokine, and a hormone.
 18. Amethod comprising increasing the relative number of hematopoietic stemcells in a cell population derived from unmobilized blood, whichpopulation includes committed cells, said hematopoietic stem cells beingcapable of being committed into more differentiated hematopoietic cells,which method comprises: (i) contacting in vivo and initial cellpopulation comprising the committed cells with an agent selected fromthe group consisting of (a) erythropoietin in combination with anantibody that binds to MHC class II antigens, and (b) GM-CSF incombination with an antibody that binds to MHC class II antigens, (ii)culturing the cell population thereby producing an altered cellpopulation comprising hematopoietic stem cells, and (iii) enriching saidhematopoietic stem cells or recovering said hematopoietic stem cellsfrom the altered cell population, wherein said hematopoietic stem cellsare enriched or recovered from the altered cell population 2 hours aftercommencement of the incubation.
 19. The method according to claim 18wherein step (iii) comprises enriching said hematopoietic stem cells orrecovering said hematopoietic stem cells from the altered cellpopulation using a cell surface marker.
 20. The method of claim 11 or18, wherein the agent comprises erythropoietin in combination with anantibody that binds to MHC class II antigens.
 21. The method of claim20, wherein the antibody that binds to MHC class II antigens is anantibody that binds to the homologous region of the beta chain of MHCclass II antigens.
 22. The method of claim 11 or 18, wherein the agentcomprises GM-CSF in combination with an antibody that binds to MHC classII antigens.
 23. The method of claim 22, wherein the antibody that bindsto MHC class II antigens is an antibody that binds to the homologousregion of the beta chain of MHC class II antigens.
 24. A methodcomprising increasing the relative number of hematopoietic stem cells ina cell population including committed cells, wherein the methodcomprises contacting in vitro a cell population derived from unmobilizedblood with an agent selected from the group consisting of (a)erythropoietin in combination with an antibody that binds to MHC classII antigens, and (b) GM-CSF in combination with an antibody that bindsto MHC class II antigens, in conjunction with cyclophosphamide, whereinthe hematopoietic stem cells are MHC class I⁺ and/or MHC class II⁺cells.